Methods, kits and antibodies for detecting parathyroid hormone

ABSTRACT

The present invention relates to novel methods and compositions useful for detecting whole parathyroid hormone at a physiological level and parathyroid fragments in a mammalian sample. Such detections may be useful to different parathyroid diseases or disorders in a subject, such as hyperparathyroidism and related bone diseases, from normal or non-disease states. One detects whole or non-fragmented (1 to 84) parathyroid hormone in a biological sample and optionally one or more of a selection of non-whole parathyroid hormone peptide fragments that may or may not function as a parathyroid hormone antagonists. By either comparing values or using independently the value of either the one or more of a selection of non-whole parathyroid hormone peptide fragments, the whole parathyroid hormone, or the combination of these values one is able to differentiate parathyroid and bone related disease states, as well as differentiate such states from normal states.

I. CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 09/344,639, filed on Jun. 26, 1999, now U.S. Pat.No. 6,743,590 B1; which is a continuation-in-part of U.S. patentapplication Ser. No. 09/231,422, filed on Jan. 14, 1999, now U.S. Pat.No. 6,689,566 B1.

II. TECHNICAL FIELD

The present invention relates to novel compositions, methods and kitsfor differentiating parathyroid diseases in a subject. Thesecompositions, methods and kits can be used, for example to differentiatehyperparathyroidism, high bone turnover, and adynamic bone disease fromnormal or non-disease states.

III. BACKGROUND OF THE INVENTION

Calcium plays an indispensable role in cell permeability, the formationof bones and teeth, blood coagulation, transmission of nerve impulse,and normal muscle contraction. The concentration of calcium ions in theblood is, along with calcitrol and calcitonin, regulated mainly byparathyroid hormone (PTH). Although calcium intake and excretion mayvary, PTH serves through a feedback mechanism to maintain a steadyconcentration of calcium in cells and surrounding fluids. When serumcalcium lowers, the parathyroid glands secrete PTH, affecting therelease of stored calcium. When serum calcium increases, stored calciumrelease is retarded through lowered secretions of PTH.

The complete form of human PTH, (hPTH), is a unique 84 amino acidpeptide (SEQ ID NO: 1), as is shown in FIG. 1. Researchers have foundthat this peptide has an anabolic effect on bone that involves a domainfor protein kinase C activation (amino acid residues 28 to 34) as wellas a domain for adenylate cyclase activation (amino acid residues 1 to7). However, various catabolic forms of clipped or fragmented PTHpeptides also are found in circulation, most likely formed byintraglandular or peripheral metabolism. For example, whole PTH can becleaved between amino acids 34 and 35 to produce a (1-34) PTH N-terminalfragment and a (35-84) PTH C-terminal fragment. Likewise, clipping canoccur between either amino acids 36 and 37 or 37 and 38. Recently, alarge PTH fragment referred to as “non-(1-84) PTH” has been disclosedwhich is clipped closer to the N-terminal end of PTH. (See LePage, R.,et al., Clin. Chem. 44: 805-810 (1998).

The clinical need for accurate measurement of PTH is well demonstrated.Serum PTH level is one of the most important index for patients with thefollowing diseases: familial hypocalciuric hypercalcemia; multipleendocrine neoplasia types I and II; osteoporosis; Paget's bone disease;primary hyperparathyroidism—caused by primary hyperplasia or adenoma ofthe parathyroid glands; pseudohypoparathyroidism; and renal failure,which can cause secondary hyperparathyroidism.

PTH plays a role in the course of disease in a patient with chronicrenal failure. Renal osteodystrophy (RO) is a complex skeletal diseasecomprising osteitis fibrosa cystica (caused by PTH excess),osteomalacia-unmineralized bone matrix (caused by vitamin D deficiency),extraskeletal calcification/ossification (caused by abnormal calcium andphosphorus metabolism), and adynamic bone disease (contributed to by PTHsuppression). Chronic renal failure patients can develop RO. Failingkidneys increase serum phosphorus (hyperphosphoremia) and decrease1,25-dihydroxyvitamin D (1,25-D) production by the kidney. The formerresults in secondary hyperparathyroidism from decreased gastrointestinalcalcium absorption and osteitis fibrosa cystica from increased PTH inresponse to an increase in serum phosphorus. The later causeshypocalcemia and osteomalacia. With the onset of secondaryhyperparathyroidism, the parathyroid gland becomes less responsive toits hormonal regulators because of decreased expression of its calciumand vitamin D receptors. Serum calcium drops. RO can lead to digitalgangrene, bone pain, bone fractures, and muscle weakness.

Determining circulating biologically active PTH levels in humans hasbeen challenging. One major problem is that PTH is found at low levels,normally 10 pg/mL to 40 pg/mL (i.e., 1 pmol/L to 4 pmol/L). Coupled withextremely low circulating levels is the problem of the heterogeneity ofPTH and its many circulating fragments. In many cases, immunoassays havefaced substantial and significant interference from circulating PTHfragments. For example, some commercially available PTH kits have almost100% cross-reactivity with the non-(1-84) PTH fragment. See the LePagearticle supra.

PTH immunoassays have varied over the years. One early approach is adouble antibody precipitation immunoassay found in U.S. Pat. No.4,369,138, issued to Arnold W. Lindall et alia. A first antibody has ahigh affinity for a (65-84) PTH fragment. A radioactive labeled (65-84)PTH peptide is added to the sample with the first antibody to competefor the unlabeled peptide. A second antibody is added which binds to anyfirst antibody and radioactive labeled PTH fragment complex, therebyforming a precipitate. Both precipitate and supernatant can be measuredfor radioactive activity, and PTH levels can be calculated therefrom.

In an effort to overcome PTH fragment interference, immunoradiometrictwo-site assays for intact PTH (I-PTH) have been introduced, such asAllegro® Intact PTH assay by the Nichols Institute of San JuanCapistrano, Calif. In one version, a capture antibody specifically bindsto the C-terminal portion of hPTH while a labeled antibody specificallybinds to the N-terminal portion of the captured hPTH. In another, twomonoclonal antibodies were used, both of which attached to theN-terminal portion of hPTH. (For the purposes of the present disclosure,the complete form of human PTH is referred to as “whole PTH” or “wPTH”as distinguished from “intact PTH” or “I-PTH” which can include not onlywPTH, but also a large PTH fragment cleaved about amino acids 5 to 8.)Unfortunately, these assays have problems in that they measure but donot discriminate between w-PTH and I-PTH. This inability comes to thefore in hyperparathyroid patients and renal failure patients who havesignificant endogenous concentrations of large, non-whole PTH fragments.

Recently, researchers have made a specific binding assay directed to thelarge N-terminal PTH fragments. See Gao, P., et al., Clinica ChimicaActa 245: 39-59 (1996). This immunochemiluminometric assay uses twomonoclonal antibodies to detect N-terminal (1-34) PTH fragments but notmid-portion PTH fragments or C-terminal PTH fragments. A key factor inthe design of these assays is to eliminate any reaction with C-terminalPTH fragments.

Nevertheless, specific whole PTH assays have not been able to measurewhole PTH at physiological levels. See, e.g., Magerlein, M., et al.,Drug Res. 48:197-204 (1998). The present invention is intended to meetthese and other needs in the art.

An important discovery leading to the present invention is that adynamicbone loses its capacity to buffer calcium and phosphate as the bones areshut down. In subjects afflicted with such conditions, they are unableto effectively buffer calcium as it enters their bodies through theirdiet. This calcium enters the blood stream and is thereafter shuttled tothe soft tissues. The parathyroid gland is particularly subject to, anddetrimentally affected by, this influx of calcium and thereby producesPTH fragments rather than, or in addition to, the active form of PTH.Accordingly, in subjects with adynamic bone, the concentration andproduction of PTH fragments is increased. In light of this and otherrelated information, the measurement of PTH fragment levels, andparticularly in conjunction with the measurement of whole PTH, can beused effectively to differentiate subjects having adynamic bone versusthose having normal bone and high bone turnover rates.

There is a tremendous need to be able to non invasively separate thedialysis patients with ADN from those suffering from high bone turnoverto avoid over treatment of ADN dialysis patients. Over treatment ofdialysis patients with ADN is a frequent occurrence under presentlyutilized methods. For example, package inserts that proscribe the use ofZemplar® and Calcijex® (Abbott Laboratories), for example, are beingused to treat thousands of dialysis patients that stand a great risk ofover treatment under the proscribed protocols that do not account forcirculating total PTH fragment levels. The present invention addressesthese and other need in the art.

IV. DISCLOSURE OF THE INVENTION

In one embodiment, the present disclosure provides an isolated antibodythat specifically binds to an N-terminal sequence of whole parathyroidhormone (PTH) and is capable of detecting said whole PTH at aphysiological level in a mammalian sample, with a proviso that saidisolated antibody avoids binding to a non-whole PTH fragment.Frequently, the isolated antibody is a monoclonal or polyclonalantibody. Also frequently, the binding between the antibody and theN-terminal sequence of whole PTH is dependent on the presence of aminoacid residues 2-5 or 3-6 of the PTH.

In one aspect an isolated antibody of the present disclosurespecifically binds to an epitope comprised in PTH₁₋₆, PTH₁₋₈, PTH₁₋₉,PTH₁₋₁₂, or PTH₃₋₁₂. Frequently, an isolated antibody of the presentdisclosure specifically binds to the parathyroid hormone peptide PTH₁₋₁₅or PTH₁₋₈, wherein at least four amino acids in said peptide sequenceare part of a reactive portion with the antibody. On occasion, anisolated antibody of the present disclosure specifically binds to anepitope comprised in PTH₁₋₅, PTH₁₋₇, PTH₁₋₈, PTH₁₋₁₀, PTH₁₋₁₁, PTH₁₋₁₃,PTH₁₋₁₄, PTH₁₋₁₅, PTH₁₋₁₆, PTH₁₋₁₇, PTH₁₋₁₈, PTH₁₋₁₉, PTH₁₋₂₀, PTH₁₋₂₁,PTH₁₋₂₂, PTH₁₋₂₃, PTH₁₋₂₄, PTH₁₋₂₅, PTH₁₋₂₆, hPTH₁₋₂₇, PTH₁₋₂₈, PTH₁₋₂₉,PTH₁₋₃₀, PTH₁₋₃₁, PTH₁₋₃₂, PTH₁₋₃₃, PTH₁₋₃₄, PTH₁₋₃₅, PTH₁₋₃₆, PTH₁₋₃₇,PTH₂₋₅, PTH₂₋₆, PTH₂₋₇, PTH₂₋₈, PTH₂₋₉, PTH₂₋₁₀, PTH₂₋₁₁, PTH₂₋₁₂,PTH₂₋₁₃, PTH₂₋₁₄, PTH₂₋₁₅, PTH₂₋₁₆, PTH₂₋₁₇, PTH₂₋₁₈, PTH₂₋₁₉, PTH₂₋₂₀,PTH₂₋₂₁, PTH₂₋₂₂, PTH₂₋₂₃, PTH₂₋₂₄, PTH₂₋₂₅, PTH₂₋₂₆, PTH₂₋₂₇, PTH₂₋₂₈,PTH₂₋₂₉, PTH₂₋₃₀, PTH₂₋₃₁, PTH₂₋₃₂, PTH₂₋₃₃, PTH₂₋₃₄, PTH₂₋₃₅, PTH₂₋₃₆,PTH₂₋₃₇, PTH₃₋₆, PTH₃₋₇, PTH₃₋₈, PTH₃₋₉, PTH₃₋₁₀, PTH₃₋₁₁, PTH₃₋₁₃,PTH₃₋₁₄, PTH₃₋₁₅, PTH₃₋₁₆, PTH₃₋₁₇, PTH₃₋₁₈, PTH₃₋₁₉, PTH₃₋₂₀, PTH₃₋₂₁,PTH₃₋₂₂, PTH₃₋₂₃, PTH₃₋₂₄, PTH₃₋₂₅, PTH₃₋₂₆, PTH₃₋₂₇, PTH₃₋₂₈, PTH₃₋₂₉,PTH₃₋₃₀, PTH₃₋₃₁, PTH₃₋₃₂, PTH₃₋₃₃, PTH₃₋₃₄, PTH₃₋₃₅, PTH₃₋₃₆, PTH₃₋₃₇,PTH₄₋₇, PTH₄₋₈, PTH₄₋₉, PTH₄₋₁₀, PTH₄₋₁₁, PTH₄₋₁₂, PTH₄₋₁₃, PTH₄₋₁₄,PTH₄₋₁₅, PTH₄₋₁₆, PTH₄₋₁₇, PTH₄₋₁₈, PTH₄₋₁₉, PTH₄₋₂₀, PTH₄₋₂₁, PTH₄₋₂₂,PTH₄₋₂₃, PTH₄₋₂₄, PTH₄₋₂₅, PTH₄₋₂₆, PTH₄₋₂₇, PTH₄₋₂₈, PTH₄₋₂₉, PTH₄₋₃₀,PTH₄₋₃₁, PTH₄₋₃₂, PTH₄₋₃₃, PTH₃₄, PTH₄₋₃₅, PTH₄₋₃₆, PTH₄ ₃₇, PTH₅₋₈,PTH₅₋₉, PTH₅₋₁₀, PTH₅₋₁₁, PTH₅₋₁₂, PTH₅₋₁₃, PTH₅₋₁₄, PTH₅₋₁₅, PTH₅₋₁₆,PTH₅₋₁₇, PTH₅₋₁₈, PTH₅₋₁₉, PTH₅₋₂₀, PTH₅₋₂₁, PTH₅₋₂₂, PTH₅₋₂₃, PTH₅₋₂₄,PTH₅₋₂₅, PTH₅₋₂₆, PTH₅₋₂₇, PTH₅₋₂₈, PTH₅₋₂₉, PTH₅₋₃₀, PTH₅₋₃₁, PTH₅₋₃₂,PTH₅₋₃₃, PTH₅₋₃₄, PTH₅₋₃₅, PTH₅₋₃₆, or PTH₅₋₃₇. Frequently, however, thenon-whole PTH fragment is a peptide having an amino acid sequence frombetween PTH₃₋₈₄ and PTH₃₄₋₈₄.

In a further embodiment a multiple antigenic peptide (MAP) is provided,which MAP comprises a branched oligolysine core conjugated with aplurality of a PTH peptide as described herein. On occasion, thebranched oligolysine core comprises 3, 7 or 15 lysine residues, also onoccasion, the MAP comprises 4, 8 or 16 copies of the PTH peptide. Theplurality of the PTH peptide comprises the same or different PTHpeptides. In one aspect, the plurality of the PTH peptide is conjugatedto the branched oligolysine core via a spacer. Frequently, the spacer isan amino acid residue. Multiple antigenic peptides comprise generallyknown technology. See, e.g., Adermann, K., et al., Innovations andPerspectives in Solid Phase Synthesis 429-32 (R. Epton, ed., MayflowerWorldwide 1994).

In another embodiment, the present disclosure provides a method formeasuring a physiological level of whole parathyroid hormone in amammalian sample, which method comprises: a) obtaining a sample from amammal to be tested; b) contacting said sample with an isolated antibodythat specifically binds to an N-terminal sequence of whole PTH and iscapable of detecting said whole PTH at a physiological level in saidmammalian sample, with a proviso that said isolated antibody avoidsbinding to a non-whole PTH fragment; and c) assessing a complex formedbetween said whole parathyroid hormone, if present in said sample, andsaid antibody, to measure physiological level of said whole parathyroidhormone in said mammalian sample. A variety of sample types may beutilized in accordance with the present methods including serum, plasmaand blood samples. Frequently, the sample is a clinical sample and themammal is a human. The non-whole PTH fragment may be any of the varietyof non-whole PTH fragments as described herein.

In one aspect, the antibody specifically binds to an epitope comprisedin PTH₁₋₆, PTH₁₋₈, PTH₁₋₉, PTH₁₋₁₂, or PTH₃₋₁₂, and/or the PTH peptidePTH₁₋₁₅. Frequently, the antibody specifically binds to a PTH epitope asdiscussed herein. On occasion, the binding between the antibody and theN-terminal sequence of whole PTH is dependent on the presence of aminoacid residues 2-5 or 3-6 of the PTH.

Although a variety of assay types are contemplated, the present methodsfrequently assess the complex formed between the whole parathyroidhormone and the antibody via a sandwich or competitive assay format. Onoccasion, the complex is assessed in a homogeneous or a heterogeneousassay format. Also frequently, the complex is assessed by a formatselected from the group consisting of an enzyme-linked immunosorbentassay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay(RIA), immunostaining, latex agglutination, indirect hemagglutinationassay (IHA), complement fixation, indirect immunofluorescent assay(IFA), nephelometry, flow cytometry assay, plasmon resonance assay,chemiluminescence assay, lateral flow immunoassay, u-capture assay,inhibition assay and avidity assay. In a sandwich assay format, theantibody that specifically binds to an N-terminal sequence of whole PTHis used as a first antibody and antibody that is capable of binding to aportion of whole PTH other than the N-terminal sequence which binds tothe first antibody is used as a second antibody. Either the firstantibody or the second antibody is frequently attached to a surface andfunctions as a capture antibody. The attachment can be direct orindirect. In a preferred embodiment, the attachment is provided via abiotin-avidin (or streptavidin) linking pair.

In another aspect, the physiological level of whole parathyroid hormoneis less than 4 pmol/L. Frequently, the physiological level of wholeparathyroid hormone is from about 0.2 pmol/L to about 4 pmol/L. Alsofrequently, the physiological range of whole PTH ranges between about 2pgm/ml to about 40 pgm/ml. On occasion, the physiological range of wholePTH ranges between about 7 pgm/ml to about 39 pgm/ml.

In a further embodiment, the present methods may be utilized to measuremultiple PTH peptide components, such as a non-whole PTH peptidefragment level and/or a total PTH level, in addition to a whole PTHlevel. In such embodiments, the methods frequently further comprisecomparing at least two parameters selected from the group consisting ofthe whole PTH level (wPTH), total PTH level, total PTH peptide fragmentlevel, C-terminal PTH fragment level (cPTH), N-terminal PTH fragmentlevel, and mid-terminal PTH fragment level (mPTH). The comparison ofparameters is generally in the form of a ratio or proportion.Frequently, the results of said comparison are used to determine whetherthe mammal, often comprising a human patient, is afflicted with a boneturnover related disorder, or used to monitor bone disease relatedtreatment. Also frequently, the present methods are used to determine ordiagnose whether the mammal is afflicted with, or at risk for, adynamicbone disease or severe hyperparathyroidism. Frequently, the presentmethods are used for clinical management of renal disease subjects andsubjects afflicted with osteoporosis, including dialysis patients. Alsofrequently, the present methods are used for diagnosing primaryhyperparathyroidism. Moreover, the present methods are useful forclinical diagnosis and management of subjects having adynamic bonedisease induced, in part, through the practice of inappropriatetreatment protocols.

In preferred embodiments of the present comparison the comparison takesmany forms. For example, the comparison can be in the form of a ratio orproportion between the whole PTH level versus the total PTH level (i.e.,represented by the equation: wPTH/total PTH); between the whole PTHlevel versus the combined cPTH and mPTH fragment levels (i.e.,represented by the equation: wPTH/(cPTH+mPTH)); between the whole PTHlevel versus the combined cPTH and mPTH fragment levels, wherein doublethe whole PTH level is subtracted from the combined cPTH and mPTHfragment levels (i.e., represented by the equation:wPTH/((cPTH−wPTH)+(mPTH−wPTH))); between the whole PTH level versus thetotal of the combined cPTH and mPTH fragment levels subtracted by thewhole PTH level (i.e., represented by the equation:wPTH/(cPTH+mPTH−wPTH)); between the whole PTH level versus the combinedwhole PTH level, cPTH and mPTH fragment levels (i.e., represented by theequation: wPTH/(wPTH+cPTH+mPTH)); between the whole PTH level versus thecPTH fragment level (i.e., represented by the equation: wPTH/cPTH);between the whole PTH level versus the mPTH fragment level (i.e.,represented by the equation: wPTH/mPTH); between the whole PTH levelversus the total PTH level minus the whole PTH level (i.e., representedby the equation: wPTH/(total PTH−wPTH)); or other combinations of thedisclosed parameters, including, without limitation, the inverse of eachcomparison. Moreover, without limitation, in one aspect, the valueobtained from determining the total PTH level and subtracting this levelfrom the whole PTH level yields the total PTH fragment level in asample/subject. The cutoff ranges for each of these comparisons as theyare associated with a particular bone turnover, treatment, disease ordisorder vary as provided herein (see e.g., Table 2 and accompanyingdiscussion).

Frequently in the present methods the sample is contacted with one ormore isolated antibodies, and wherein each of said one or more isolatedantibodies specifically binds one or more PTH peptide fragments selectedfrom the group consisting of: PTH₃₉₋₈₄, PTH₁₋₃₄, PTH₄₃₋₆₈, PTH₇₋₈₄,PTH₃₉₋₆₈, PTH₅₃₋₈₄, PTH₆₅₋₈₄, PTH₄₄₋₆₈, PTH₁₉₋₈₄, PTH₂₃₋₈₄, PTH₁₋₃₈,PTH₁₋₄₈, PTH₁₋₅₈, PTH₁₋₆₈, and PTH₁₋₇₈.

The present methods of measuring multiple PTH components provide avariety of uses. For example, such methods are used for differentiatingbetween a person having substantially normal parathyroid function andhaving hyperparathyroidism, e.g., primary hyperparathyroidism;monitoring parathyroid related bone disease and treatment; monitoringeffects of therapeutic treatment for hyperparathyroidism; diagnosingparathyroid related bone disease; clinical management of renal diseasesubjects and renal disease related treatments and subjects afflictedwith osteoporosis and osteoporosis related treatments.

The present disclosure further provides kits for carrying out thepresently described methods and utilizing the peptides and antibodies asdescribed herein. In one embodiment, a kit for measuring a physiologicallevel of whole parathyroid hormone in a mammalian sample, which kitcomprises, in a container, an isolated antibody that specifically bindsto an N-terminal sequence of whole parathyroid hormone (PTH) and iscapable of detecting said whole PTH at a physiological level in amammalian sample, with a proviso that said isolated antibody avoidsbinding to a non-whole PTH fragment.

In another embodiment, the present disclosure further provides kits forproducing an antibody to a parathyroid hormone (PTH) or a PTH peptide,which kits comprise: a) an isolated PTH peptide; b) means forintroducing said isolated PTH peptide to a mammal in an amountsufficient to produce an antibody to said PTH peptide; and c) means forrecovering said antibody from said mammal. In further embodiment, a kitfor producing an antibody to a parathyroid hormone (PTH) or a PTHpeptide is provided which comprises: a) a MAP; b) a means forintroducing said MAP to a mammal in an amount sufficient to produce anantibody to a PTH peptide comprised in said MAP; and b) a means forrecovering said antibody from said mammal. In a still furtherembodiment, a kit is provided for producing an antibody to a parathyroidhormone (PTH) or a PTH peptide, which kit comprises: a) a PTH protein orpeptide from between PTH₁₋₃₄ and PTH₁₋₈₄; b) means for introducing saidPTH protein or peptide from between PTH₁₋₃₄ and PTH₁₋₈₄ to a mammal inan amount sufficient to produce an antibody to said PTH protein orpeptide; c) means for recovering said antibody from said mammal; and c)another specific PTH peptide.

An isolated PTH peptide in such kits can be any of the variety of PTHpeptides as described herein. Frequently, the PTH peptide are conjugatedto a carrier to enhance the PTH peptide's immunogenecity, e.g., acarrier protein, which may together form a fusion protein. For example,such PTH peptide is selected from the group consisting of PTH₁₋₁₁,PTH₁₋₁₃, PTH₁₋₁₄, PTH₁₋₁₅, PTH₁₋₁₆, PTH₁₋₁₇, PTH₁₋₁₈, PTH₁₋₁₉, PTH₁₋₂₀,PTH₁₋₂₁, PTH₁₋₂₂, PTH₁₋₂₃, PTH₁₋₂₄, PTH₁₋₂₅, PTH₁₋₂₆, hPTH₁₋₂₇, PTH₁₋₂₈,PTH₁₋₂₉, PTH₁₋₃₀, PTH₁₋₃₁, PTH₁₋₃₂, PTH₁₋₃₃, PTH₁₋₃₄, PTH₁₋₃₅, PTH₁₋₃₆,PTH₂₋₅, PTH₂₋₆, PTH₂₋₈, PTH₂₋₉, PTH₂₋₁₀, PTH₂₋₁₁, PTH₂₋₁₂, PTH₂₋₁₃,PTH₂₋₁₄, PTH₂₋₁₅, PTH₂₋₁₆, PTH₂₋₁₇, PTH₂₋₁₈, PTH₂₋₁₉, PTH₂₋₂₀, PTH₂₋₂₁,PTH₂₋₂₂, PTH₂₋₂₃, PTH₂₋₂₄, PTH₂₋₂₅, PTH₂₋₂₆, PTH₂₋₂₇, PTH₂₋₂₈, PTH₂₋₂₉,PTH₂₋₃₀, PTH₂₋₃₁, PTH₂₋₃₂, PTH₂₋₃₃, PTH₂₋₃₄, PTH₂₋₃₅, PTH₂₋₃₆, PTH₃₋₆,PTH₃₋₇, PTH₃₋₉, PTH₃₋₁₀, PTH₃₋₁₁, PTH₃₋₁₂, PTH₃₋₁₃, PTH₃₋₁₄, PTH₃₋₁₅,PTH₃₋₁₆, PTH₃₋₁₇, PTH₃₋₁₈, PTH₃₋₁₉, PTH₃₋₂₀, PTH₃₋₂₁, PTH₃₋₂₂, PTH₃₋₂₃,PTH₃₋₂₄, PTH₃₋₂₅, PTH₃₋₂₆, PTH₃₋₂₇, PTH₃₋₂₈, PTH₃₋₂₉, PTH₃₋₃₀, PTH₃₋₃₁,PTH₃₋₃₂, PTH₃₋₃₃, PTH₃₋₃₄, PTH₃₋₃₅, PTH₃₋₃₆, PTH₄₋₇, PTH₄₋₈, PTH₄₋₉,PTH₄₋₁₀, PTH₄₋₁₁, PTH₄₋₁₃, PTH₄₋₁₄, PTH₄₋₁₅, PTH₄₋₁₆, PTH₄₋₁₇, PTH₄₋₁₈,PTH₄₋₁₉, PTH₄₋₂₀, PTH₄₋₂₁, PTH₄₋₂₂, PTH₄₋₂₃, PTH₄₋₂₄, PTH₄₋₂₅, PTH₄₋₂₆,PTH₄₋₂₇, PTH₄₋₂₈, PTH₄₋₂₉, PTH₄₋₃₀, PTH₄₋₃₁, PTH₄₋₃₂, PTH₄₋₃₃, PTH₄₋₃₄,PTH₄₋₃₅, PTH₄₋₃₆, PTH₅₋₈, PTH₅₋₉, PTH₅₋₁₁, PTH₅₋₁₂, PTH₅₋₁₃, PTH₅₋₁₄,PTH₅₋₁₅, PTH₅₋₁₆, PTH₅₋₁₇, PTH₅₋₁₈, PTH₅₋₁₉, PTH₅₋₂₀, PTH₅₋₂₁, PTH₅₋₂₂,PTH₅₋₂₃, PTH₅₋₂₄, PTH₅₋₂₅, PTH₅₋₂₆, PTH₅₋₂₇, PTH₅₋₂₈, PTH₅₋₂₉, PTH₅₋₃₀,PTH₅₋₃₁, PTH₅₋₃₂, PTH₅₋₃₃, PTH₅₋₃₄, PTH₅₋₃₅, PTH₅₋₃₆, and PTH₅₋₃₇.

The presently contemplated kits may also provide an immunogen comprisinga PTH peptide as described herein, together with an immune responsepotentiator. On occasion, the immune response potentiator is selectedfrom the group consisting of Bacille Calmette-Guerin (BCG),Corynebacterium Parvum, Brucella abortus extract, glucan, levamisole,tilorone, an enzyme and a non-virulent virus.

The present disclosure further provides methods for producing anantibody to a parathyroid hormone (PTH) or a PTH peptide. In oneembodiment, such method comprises: a) introducing an isolated PTHpeptide to a mammal in an amount sufficient to produce an antibody tosaid PTH peptide; and b) recovering said antibody from said mammal.Another frequent method for producing an antibody to a parathyroidhormone (PTH) or a PTH peptide comprises: a) introducing a MAP to amammal in an amount sufficient to produce an antibody to a PTH peptidecomprised in said MAP; and b) recovering said antibody from said mammal.In one aspect, the present disclosure provides antibodies to a PTH or aPTH peptide produced by these methods. In a related embodiment, a methodis provided for producing an antibody to a parathyroid hormone (PTH) ora PTH peptide, which method comprises: a) introducing a PTH protein orpeptide from between PTH₁₋₃₄ and PTH₁₋₈₄ to a mammal in an amountsufficient to produce an antibody to said PTH protein or peptide; b)recovering said antibody from said mammal; and c) affinity purifying aPTH antibody that specifically binds to an epitope comprised in a PTHpeptide using said PTH peptide. In a further embodiment, the presentdisclosure provides an antibody to a PTH or a PTH peptide produced bysuch methods.

V. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic view of whole human PTH (SEQ ID NO: 1).

FIG. 2 is a diagrammatic view of a wPTH assay using the present antibodyas a tracer element.

FIG. 3 is a diagrammatic view of a wPTH assay using the present antibodyas a capture element.

FIG. 4 is a graph showing a standard curve for a wPTH assay.

FIG. 5 is a graph comparing a conventional I-PTH assay with the presentwPTH assay for healthy normal persons with “normal” PTH values.

FIG. 6 is a diagrammatic view showing interference from non (1-84) PTHfragments in conventional I-PTH assays.

FIG. 7 is a graph comparing a conventional I-PTH assay with the presentwPTH assay for patients with chronic uremia.

FIG. 8 is a graph showing the distribution of values for healthy normalpersons, patients with primary hyperparathyroidism, and patients withchronic uremia.

FIG. 9 is a diagrammatic view showing how PIN blocks the action of wPTHat the receptor level, thereby making the person insensitive to thebiological effects of wPTH.

FIG. 10 is a graph demonstrating complete cross-reactivity of wPTH andPIN in a total PTH assay used in the present invention.

FIG. 11 is a graph demonstrating how the whole PTH assay used in thepresent invention does not detect to PIN.

FIG. 12 is a graph demonstrating how PIN is an in vivo inhibitor ofwPTH.

FIG. 13 illustrates comparison of the recognition of hPTH 1-84 and hPTH7-84 by the Nichols I-PTH assay. The Nichols I-PTH assay does notdifferentiate between hPTH 1-84 (solid line) and hPTH 7-84 (dashedline).

FIG. 14 illustrates comparison of the recognition of hPTH 1-84 and hPTH7-84 by the Whole PTH assay. Unlike the Nichols I-PTH assay, the WholePTH assay does discriminate between hPTH 1-84 (solid line) and hPTH 7-84(dashed line). Concentrations of hPTH 7-84 as high as 10,000 pg wereundetectable.

FIG. 15 illustrates comparison of the effect of hPTH 1-84 or hPTH 7-84on cAMP production in ROS 17.2 cells. Unlike hPTH 7-84, hPTH 1-84increased cAMP production in a dose-dependent manner. cAMP increasedfrom 18.1±1.2 to 738±4.1 nmol/well after treatment with 10⁻⁸ mol/L hPTH1-84. The same concentration of hPTH 7-84 had no effect.

FIG. 16 illustrates comparison of the effects of hPTH 1-84 or hPTH 1-84plus hPTH 7-84 on (A) glomerular filtration rate (GFR) and (B)fractional excretion of phosphorus (FE_(po4)). Control and treatmentperiods are denoted by open and closed bars, respectively. Thephosphaturia induced by hPTH 1-84 was decreased by 50.2% (P<0.05) whenanimals were treated simultaneously with 7-84 PTH, despite a significantincrease in GFR (P<0.005).

FIG. 17 illustrates comparison of PTH values in plasma from uremicpatients using the Nichols “intact” PTH assay (▪) versus the Whole PTHassay (●). Plasma PTH values are uniformly higher when measured with theNichols “intact” PTH assay than with the Whole PTH assay. The median PTHvalues were 523 vs. 344 pg/mL, respectively (P<0.001).

FIG. 18 illustrates effects of plasma calcium on PTH degradation indialysis patients. The percentage of non-(1-84) PTH fragment (likelyhPTH 7-84) correlates positively with plasma calcium (P<0.02) (r=0.638;P=0.0025; N=20).

FIG. 19 illustrates comparison of plasma PTH levels in renal transplantpatients using Nichols I-PTH and Whole PTH assays. PTH values are higherwhen measured with the Nichols I-PTH assay (P>0.005).

FIG. 20 illustrates intracellular PTH content on parathyroid glands fromuremic patients. The 41.8±3.2% of the total PTH, measured by theI-Nichols assay (expressed as 100%), represents the non-(1-84) PTHfragment “likely” hPTH 7-84 (□). the 1-84 PTH molecule was measured withthe Whole PTH assay (▪).

VI. DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, patentapplications (published or unpublished), and other publications referredto herein are incorporated by reference in their entirety. If adefinition set forth in this section is contrary to or otherwiseinconsistent with a definition set forth in the patents, applications,published applications and other publications that are hereinincorporated by reference, the definition set forth in this sectionprevails over the definition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, “antibody” is used in the broadest sense. Therefore, an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology and/or afunctional fragment thereof. Antibodies of the present inventioncomprise monoclonal and polyclonal antibodies as well as fragmentscontaining the antigen-binding domain and/or one or more complementaritydetermining regions of these antibodies.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theantibodies comprising the population are identical except for possiblenaturally occurring mutations that are present in minor amounts. As usedherein, a “monoclonal antibody” further refers to functional fragmentsof monoclonal antibodies.

As used herein, “mammal” refers to any of the mammalian class ofspecies. Frequently, the term “mammal,” as used herein, refers tohumans, human subjects or human patients.

As used herein, “whole parathyroid hormone (PTH)” or “wPTH” refers tothe complete molecule of PTH. This term is not species-specific unlessotherwise designated. For purposes herein, the name “parathyroid hormone(PTH)” is used herein, although all other names are contemplated. It isintended to encompass whole PTH with conservative amino acidsubstitutions that do not substantially alter its biological activity.Suitable conservative substitutions of amino acids are known to those ofskill in this art and may be made generally without altering thebiological activity of the resulting molecule. Those of skill in thisart recognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson et al., MOLECULAR BIOLOGY OF THEGENE, 4th Edition, 1987, The Bejamin/Cummings Pub. Co., p.224).

As used herein, “parathyroid hormone (PTH) agonist,” “cyclase activatingPTH” or “CAP” refers to the complete molecule of PTH or a fragment,derivative or analog thereof that stimulates osteoclasts formation andbone turnover to increase blood calcium levels. PTH agonist furtherrefers to peptides which have PTH agonist properties. Other names of PTHinclude parathormone and parathyrin. For purposes herein, the name“parathyroid hormone (PTH)” is used herein, although all other names arecontemplated. It is intended to encompass PTH agonist with conservativeamino acid substitutions that do not substantially alter its biologicalactivity. Suitable conservative substitutions of amino acids are knownto those of skill in this art and may be made generally without alteringthe biological activity of the resulting molecule. Those of skill inthis art recognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson et al., MOLECULAR BIOLOGY OF THEGENE, 4th Edition, 1987, The Bejamin/Cummings Pub. co., p.224). PTHagonist assay values may be obtained by measuring a sample with aScantibodies Whole PTH Assay or a Scantibodies CAP Assay or a 3^(rd)generation PTH Assay or a Nichols BioIntact PTH assay or an ImmutopicsHuman Bioactive PTH assay.

As used herein, the term “total PTH” refers to a total accounting ofwhole PTH levels in addition to PTH fragment levels. Moreover, this termis not species-specific unless otherwise designated.

As used herein, the term “PIN” refers to PTH fragments that have PTHantagonistic or inhibiting properties. Therefore, although occasionallyof concurrent scope, a reference to PTH fragments, as provided herein,is not intended to be limited to PIN.

As used herein, a “PTH fragment” is a PTH peptide that comprises anon-whole contiguous portion of an entire PTH protein. A reference to aPTH fragment as herein includes C-terminal, mid-terminal fragments andPIN, unless otherwise indicated. Moreover, this term is notspecies-specific unless otherwise designated.

As used herein, “treatment” means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Treatment also encompasses any pharmaceutical use of thecompositions herein.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from, e.g., infection or genetic defect, andcharacterized by identifiable symptoms.

As used herein, “high bone turnover” refers to the bone turnover rate asbeing above a normal bone turnover rate in a subject and is one of thesymptoms manifested in subjects having hyperparathyroidism. While notbound by theory, a subject afflicted with severe hyperparathyroidism hasa higher bone turnover rate than the same subject afflicted with mildhyperparathoidism, however, both having a high bone turnover rate ascompared with a normal subject and a subject afflicted with adynamicbone disease.

As used herein, the term “subject” is not limited to a specific speciesor sample type. For example, the term “subject” may refer to a patient,and frequently a human patient. However, this term is not limited tohumans and thus encompasses a variety of mammalian species.

As used herein, “afflicted” as it relates to a disease or disorderrefers to a subject having or directly affected by the designateddisease or disorder.

As used herein the term “sample” refers to anything which may contain ananalyte for which an analyte assay is desired. The sample may be abiological sample, such as a biological fluid or a biological tissue.Examples of biological fluids include urine, blood, plasma, serum,saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus,amniotic fluid or the like. Biological tissues are aggregate of cells,usually of a particular kind together with their intercellular substancethat form one of the structural materials of a human, animal, plant,bacterial, fungal or viral structure, including connective, epithelium,muscle and nerve tissues. Examples of biological tissues also includeorgans, tumors, lymph nodes, arteries and individual cell(s).

As used herein the term “avoids binding” refers to the specificity ofparticular antibodies or antibody fragments. Antibodies or antibodyfragments that avoid binding a particular moiety generally contain aspecificity such that a large percentage of the particular moiety wouldnot be bound by such antibodies or antibody fragments. This percentagegenerally lies within the acceptable cross reactivity percentage withinterfering moieties of assays utilizing antibodies directed todetecting a specific target. Frequently, antibodies or antibodyfragments of the present disclosure avoid binding greater than about 90%of an interfering moiety, although higher percentages are clearlycontemplated and preferred. For example, antibodies or antibodyfragments of the present disclosure avoid binding about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, and about 99% or more of an interfering moiety. Less occasionally,antibodies or antibody fragments of the present disclosure avoid bindinggreater than about 70%, or greater than about 75%, or greater than about80%, or greater than about 85% of an interfering moiety. Although notbound by theory, as contemplated herein, an interfering moiety maycomprise a non-whole PTH fragment.

As used herein the term “physiological level of whole PTH” refersgenerally to the average concentration of whole PTH present in a mammal,e.g., a human, expressed in pmol/L, or another suitable measurement unit(e.g., pgm/ml). See, e.g., Woodhead, J. S., Clin. Biochem. 23, 17(1990). In one aspect, the physiological range of whole PTH rangesbetween about 0.2 pmol/L to about 4 pmol/L, or about 2 pgm/ml to about40 pgm/ml. On occasion, the physiological range of whole PTH can rangebetween about 7 pgm/ml to about 39 pgm/ml. Although specific ranges aredescribed herein as representative of a physiological range, one ofskill in the art would understand that the physiological level of wholePTH may lie outside of the presently disclosed ranges in certainsubjects. Nevertheless, the compositions and methods provided herein areuseful to detect discreet concentrations of whole PTH and havesensitivities within the physiological range as provided herein.

As used herein, the term “N-terminal” refers to the amino terminus of aPTH polypeptide having a free amino group. With reference to a PTHfragment, an N-terminal PTH fragment refers to a non-whole contiguousportion of PTH having an intact N-terminal. An “intact N-terminal” asused herein refers to PTH or a PTH fragment having an intact 1stposition of PTH₁₋₈₄. This first position is also referred to herein asan “original N-terminus” or an “original N-terminal.”

As used herein, the term “C-terminal” refers to the carboxyl terminus ofa PTH polypeptide having a free carboxyl group. With reference to a PTHfragment, a C-terminal PTH fragment refers to a non-whole contiguousportion of PTH having an intact C-terminal. An “intact C-terminal” asused herein refers to PTH or a PTH fragment having an intact 84thposition of PTH₁₋₈₄. This 84th position is also referred to herein as an“original C-terminus” or an “original C-terminal.”

As used herein, the term “mid-terminal PTH fragment” refers to anon-whole contiguous portion of PTH having neither an intact N-terminalnor an intact C-terminal. These types of PTH fragments may also bereferred to herein as “mid-terminus fragments.”

As used herein, the term “specifically binds” refers to the specificityof an antibody such that it preferentially binds to a defined target.Recognition by an antibody of a particular target in the presence ofother potential targets is one characteristic of such binding. Specificbinding of the presently contemplated antibodies to particular PTHtargets is measured through known methods utilizing the tools providedherein.

As used herein, “stringency” of hybridization reactions is readilydeterminable by one of ordinary skill in the art, and generally is anempirical calculation dependent upon probe length, washing temperature,and salt concentration. In general, longer probes require highertemperatures for proper annealing, while shorter probes need lowertemperatures. Hybridization generally depends on the ability ofdenatured nucleic acid sequences to reanneal when complementary strandsare present in an environment below their melting temperature. Thehigher the degree of desired homology between the probe and hybridizablesequence, the higher the relative temperature that can be used. As aresult, it follows that higher relative temperatures would tend to makethe reaction conditions more stringent, while lower temperatures lessso. For additional details and explanation of stringency ofhybridization reactions, see Current Protocols in Molecular Biology(Ausubel et al. eds., Wiley Interscience Publishers, 1995); MolecularCloning: A Laboratory Manual (J. Sambrook, E. Fritsch, T. Maniatis eds.,Cold Spring Harbor Laboratory Press, 2d ed. 1989); Wood et al., Proc.Natl. Acad. Sci. USA, 82:1585-1588 (1985).

As used herein the term “isolated” refers to material removed from itsoriginal environment, and is altered from its natural state. Forexample, an isolated polypeptide could be coupled to a carrier, andstill be “isolated” because that polypeptide is not in its originalenvironment.

The present disclosure encompasses antigens, antibodies and methods ofproducing antibodies that have a particular specificity to targetproteins and/or peptides which contain a specific amino acid residue ormultiple amino acid residues, in a series or otherwise. The specificamino acid residue(s) may be located in the N-terminal region of aproteins or peptide or in the C-terminal region. Moreover the specificamino acid residue(s) may be located in a region between the N-terminaland C-terminal regions of a protein or peptide. Occasionally, when thereis more than one specific amino acid residue, such residues may bedispersed in any one or more of the N-terminal, C-terminal, betweenthese two regions, and/or in all of these regions.

In disclosing the present invention, one should remember that there area number of closely analogous, species dependent forms of PTH. The aminoacid sequence of hPTH is shown in FIG. 1. However, for rat PTH, mousePTH, bovine PTH, canine PTH, horse PTH or porcine PTH, for example, onefinds the substitutions at some of the amino acids in the hPTH sequence.For the purposes of the present invention, one can use interchangeablyantibodies or antibody fragments to forms of these PTHs, although it ispreferred to use an antibody with specificity for PTH having a sequencematching the species in which the PTH measurements are made.

B. Parathyroid Hormone Fragments

In general, a PTH fragment of the present invention comprises anon-whole contiguous portion of PTH having an amino acid sequence as setforth in SEQ ID NOs: 1 2, 3, 4, 5, 6, and/or 7 (PTH₁₋₈₄), or a nucleicacid encoding said portion of PTH. A PTH fragment may have the followingcharacteristics: a) the N-terminal amino acid residue of said PTHfragment starts at any position spanning position 1 through position 80of said PTH₁₋₈₄; b) the C-terminal amino acid residue of said PTHfragment ends at any position spanning position 4 through position 84 ofsaid PTH₁₋₈₄; and c) said PTH fragment has a minimal length of threeamino acid residues. Preferably, the PTH fragment is in the form of apharmaceutical composition.

PTH fragments of the present invention are organized into threecategories: N-terminal, C-terminal, and mid-terminal PTH fragments. Asfurther described herein, N-terminal fragments comprise a non-wholecontiguous portion of PTH having an intact N-terminus, but not an intactoriginal C-terminus. As also described herein, C-terminal fragmentscomprise a non-whole contiguous portion of PTH having an intactC-terminus, but not an intact original N-terminus. Moreover, as furtherdescribed herein, mid-terminal fragments comprise a non-whole contiguousportion of PTH having neither an intact original C-terminus, nor intactoriginal N-terminus. All mammalian sources/sequences of PTH arecontemplated.

In one embodiment, PTH fragments comprise a subset of cyclase inactivePTH. However, in light of the present description, a variety of otherPTH fragments are contemplated, ascertainable and useful in the presentcompositions, kits and methods. Importantly, PTH₇₋₈₄ represents a memberof the group of PTH fragments currently contemplated. The presentdisclosure further contemplates large inactive PTH fragments in thedescription of PTH fragments.

In one embodiment, the N-terminal amino acid residue of the PTH fragmentstarts at any defined position spanning position 2 through position 70of said PTH₁₋₈₄. The C-terminal amino acid residue of said PTH fragmentends at any defined position spanning position 35 through position 84 ofsaid PTH₁₋₈₄. Therefore, for example, fragments ranging from PTH₂₋₈₄ toPTH₃₄₋₈₄ to PTH₇₀₋₈₄ are included as C-terminal fragments. Mid-terminalPTH fragments are also contemplated, for example, ranging withinPTH₃₉₋₆₈ or PTH₄₄₋₆₈. For example, mid-terminal PTH fragments havingtheir N-terminal beginning around position 44 of said PTH₁₋₈₄ and theirC-terminal ending around position 68 of said PTH₁₋₈₄ are included in thepresent description. Without being bound by theory, a mid-terminal PTHfragment does not include position 1, nor position 84 of said PTH₁₋₈₄,but rather falls within these positions.

In a specific embodiment, the PTH fragment is a protein or a peptide, ora nucleic acid encoding said protein or peptide, selected from the groupconsisting of PTH₂₋₈₄, PTH₃₋₈₄, PTH₄₋₈₄, PTH₅₋₈₄, PTH₆₋₈₄, PTH₇₋₈₄,PTH₈₋₈₄, PTH₉₋₈₄, PTH₁₀₋₈₄, PTH₁₁₋₈₄, PTH₁₂₋₈₄, PTH₁₃₋₈₄, PTH₁₄₋₈₄,PTH₁₅₋₈₄, PTH₁₆₋₈₄, PTH₁₇₋₈₄, PTH₁₈₋₈₄, PTH₁₉₋₈₄, PTH₂₀₋₈₄, PTH₂₁₋₈₄,PTH₂₂₋₈₄, PTH₂₃₋₈₄, PTH₂₄₋₈₄, PTH₂₅₋₈₄, PTH₂₆₋₈₄, PTH₂₇₋₈₄, PTH₂₈₋₈₄,PTH₂₉₋₈₄, PTH₃₀₋₈₄, PTH₃₁₋₈₄, PTH₃₂₋₈₄, and PTH₃₃₋₈₄. In anotherspecific embodiment, the PTH fragment is a protein or a peptide, or anucleic acid encoding said protein or peptide, selected from the groupconsisting of PTH₇₋₆₉, PTH₇₋₇₀, PTH₇₋₇₁, PTH₇₋₇₂, PTH₇₋₇₃, PTH₇₋₇₄,PTH₇₋₇₅, PTH₇₋₇₆, PTH₇₋₇₇, PTH₇₋₇₈, PTH₇₋₇₉, PTH₇₋₈₀, PTH₇₋₈₁, PTH₇₋₈₂,PTH₇₋₈₃ and PTH₇₋₈₄.

In another embodiment, the PTH fragment is a protein or a peptide, or anucleic acid encoding said protein or peptide, selected from the groupconsisting of PTH₃₄₋₈₄, PTH₃₅₋₈₄, PTH₃₆₋₈₄, PTH₃₇₋₈₄, PTH₃₈₋₈₄,PTH₃₉₋₈₄, PTH₄₀₋₈₄, PTH₄₁₋₈₄, PTH₄₂₋₈₄, PTH₄₃₋₈₄, PTH₄₄₋₈₄, PTH₄₅₋₈₄,PTH₄₆₋₈₄, PTH₄₇₋₈₄, PTH₄₈₋₈₄, PTH₄₉₋₈₄, PTH₅₀₋₈₄, PTH₅₁₋₈₄, PTH₅₂₋₈₄,PTH₅₃₋₈₄, PTH₅₄₋₈₄, PTH₅₅₋₈₄, PTH₅₆₋₈₄, PTH₅₇₋₈₄, PTH₅₈₋₈₄, PTH₅₉₋₈₄,PTH₆₀₋₈₄, PTH₆₁₋₈₄, PTH₆₂₋₈₄, PTH₆₃₋₈₄, PTH₆₄₋₈₄, PTH₆₅₋₈₄, PTH₆₆₋₈₄,PTH₆₇₋₈₄, PTH₆₈₋₈₄, PTH₆₉₋₈₄, and PTH₇₀₋₈₄.

In a further embodiment, the PTH fragment is a protein or a peptide, ora nucleic acid encoding said protein or peptide, selected from the groupconsisting of PTH₇₋₆₀, PTH₈₋₆₀, PTH₉₋₆₀, PTH ₁₀₋₆₀, PTH₁₁₋₆₀, PTH₁₂₋₆₀,PTH₁₃₋₆₀, PTH₁₄₋₆₀, PTH₁₅₋₆₀, PTH₁₆₋₆₀, PTH ₁₇₋₆₀, PTH₁₈₋₆₀, PTH₁₉₋₆₀,PTH₂₀₋₆₀, PTH₂₁₋₆₀, PTH₂₂₋₆₀, PTH₂₃₋₆₀, PTH₂₄₋₆₀, PTH₂₅₋₆₀, PTH₂₆₋₄₀,PTH₂₇₋₆₀, PTH₂₈₋₆₀, PTH₂₉₋₆₀, PTH₃₀₋₆₀, PTH₃₁₋₆₀, PTH₃₂₋₆₀, PTH₃₃₋₆₀,PTH₃₄₋₆₀, PTH₃₅₋₆₀, PTH₃₆₋₆₀, PTH₃₇₋₆₀, and PTH₃₈₋₆₀, PTH₃₉₋₆₀,PTH₄₀₋₆₀, PTH₄₁₋₆₀, PTH₄₂₋₆₀, PTH₄₃₋₆₀, PTH₄₄₋₅₉, PTH₄₄₋₆₀, PTH₄₅₋₆₀,PTH₄₆₋₆₀, PTH₄₇₋₆₀, and PTH₄₈₋₆₀, and other mid-terminal PTH fragmentsas described herein. In another specific embodiment, the PTH fragment isa protein or a peptide, or a nucleic acid encoding said protein orpeptide, selected from the group consisting of PTH₁₃₋₃₄, PTH₇₋₅₃,PTH₈₋₅₃, PTH₉₋₅₃, PTH₁₀₋₅₃, PTH₁₁₋₅₃, PTH₁₂₋₅₃, PTH₁₃₋₅₃, PTH₁₄₋₅₃,PTH₁₅₋₅₃, PTH₁₆₋₅₃, PTH₇₋₅₃, PTH₁₈₋₅₃, PTH₁₉₋₅₃, PTH₂₀₋₅₃, PTH₂₁₋₅₃,PTH₂₂₋₅₃, PTH₂₃₋₅₃, PTH₂₄₋₅₃, PTH₂₅₋₅₃, PTH₂₆₋₅₃, PTH₂₇₋₅₃, PTH₂₈₋₅₃,PTH₂₉₋₅₃, PTH₃₀₋₅₃, PTH₃₁₋₅₃, PTH₃₂₋₅₃, PTH₃₃₋₅₃, PTH₃₄₋₅₃, PTH₃₅₋₅₃,PTH₃₆₋₅₃, PTH₃₇₋₅₃, and PTH₃₈₋₅₃, and other mid-terminal PTH fragmentsas described herein.

In another preferred embodiment, a PTH fragment comprises, or anantibody specifically binds, a PTH peptide fragment selected from thegroup consisting of: PTH₃₉₋₈₄, PTH₁₋₃₄, PTH₄₃₋₆₈, PTH₇₋₈₄, PTH₃₉₋₆₈,PTH₅₃₋₈₄, PTH₆₅₋₈₄, PTH₄₄₋₆₈, PTH₁₉₋₈₄, PTH₂₃₋₈₄, PTH₁₋₆₈, or acombination of two or more from this group. This group may furthercomprise a PTH peptide fragment having an N-terminus starting betweenposition 39 to 65 of PTH₁₋₈₄, and having a C-terminal position ending atposition 84 of PTH₁₋₈₄. In a particularly preferred embodiment, a PTHpeptide fragment comprises a PTH fragment present and detectable innature.

In another embodiment, the PTH fragment is a protein or a peptide, or anucleic acid encoding said protein or peptide having an intactN-terminus, for example, without limitation, PTH₁₋₃₈, PTH₁₋₄₈, PTH₁₋₅₈,PTH₁₋₆₈, PTH₇₈, amongst other intact N-terminal PTH fragments.

The PTH fragment can have any suitable length and may have PTH agonizingor antagonizing activity, although PTH agonizing or antagonizingactivity is not required of the present PTH fragments. For example, thePTH fragment can have a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 or 83 aminoacid residues.

C. PTH Ratios—Whole PTH and PTH Fragments

An important discovery leading to the present invention is that adynamicbone loses its capacity to buffer calcium and phosphate as the bones areshut down. In subjects afflicted with such conditions, they are unableto effectively buffer calcium as it enters their bodies through theirdiet. This calcium enters the blood stream and is thereafter shuttled tothe soft tissues. The parathyroid gland is particularly subject to, anddetrimentally affected by, this influx of calcium and thereby producesPTH fragments rather than, or in addition to, the active form of PTH.See, e.g., Mayer GP, et al., Endocrinology 104: 1778-1784 (1979);D'Amour P, et al., J. Clin. Endocrinol. Metab. 74: 525-532 (1992);D'Amour P, et al., J. Bone Miner. Res. 11: 1075-1085 (1996); Cardinal,H., et al., J. Clin. Endocrinol. Metab. 83: 3839-44 (1998). Accordingly,in subjects with adynamic bone, the concentration and production of PTHfragments is increased. In light of this and other related information,the measurement of PTH fragment levels, and particularly in conjunctionwith the measurement of whole PTH, can be used effectively todifferentiate subjects having adynamic bone versus those having normalbone and high bone turnover rates.

The present disclosure includes these findings in the presentation ofpeptides, antibodies, methods and kits for the measurement of PTHlevels. In one preferred embodiment, the present methods utilize a ratioof whole PTH to total PTH, wherein the total PTH level comprises wholePTH plus PTH fragments in addition to PTH₇₋₈₄, such as other PTHfragments described herein (i.e., whole PTH/total PTH ratio). This ratiocontains an increased total PTH concentration as compared with a PTHratio that comprises a total PTH level reflecting measurement of wholePTH plus PTH fragments that specifically bind to an antibody generatedfrom PTH₇₋₈₄ peptide. Accordingly, the CAP/PTH fragment ratio accordingis generally lower than the CAP/(CAP+PTH₇₋₈₄) ratio. Until presently,recognition of the predictive and therapeutic benefits of PTH ratioshave been unrecognized. See, e.g., Martine-Esther Cohen Solal, et al., JClin. Endocrinol. Metab. 73: 516-524 (1991) (concluding that themeasurement of whole PTH is “superior to C-terminal and midregion assaysfor the prediction of histological type bone diseases.”).

In one embodiment, a total PTH assay is utilized wherein an antibodyspecific for PTH₄₄₋₆₈ is utilized in addition to an antibody specificfor PTH₅₂₋₈₄ in addition to other potential antibodies to determine atotal PTH level.

The present disclosure also provides a more therapeutically predictivePTH ratios involving whole PTH levels, PTH fragment levels, andoccasionally total PTH levels. The total PTH levels in this ratioinclude PTH fragments in addition to large PTH N-terminal fragments suchas PTH₇₋₈₄. These PTH fragments include a category of PTH fragmentsreferred to elsewhere in the present disclosure as large inactivefragments and are not necessarily C-terminal nor N-terminal PTHfragments as described herein. As provided herein, PTH fragmentsincluded in the present total PTH determinations include PTH₇₋₈₄, inaddition to other PTH fragments. Thus, an important aspect of thepresent enhanced PTH ratio involves monitoring a majority, and morepreferably all, of the circulating PTH fragments in a subject such thata total PTH assay will measure N-terminal, C-terminal and mid-terminalPTH fragments. In a related aspect, the fragments measured in theenhanced ratio include PTH₇₋₈₄ in addition to other PTH fragments asdescribed herein.

For example, an enhanced PTH ratio comprised of whole PTH versus totalPTH (measuring most or all circulating PTH fragments in addition towhole PTH) will generally provide a lower percentage as compared with aPTH ratio measuring a total PTH ratio comprising whole PTH versus“total” PTH (consisting of whole PTH plus large N-terminal PTH fragmentssuch as PTH₇₋₈₄). This lower percentage is due to the measurement ofadditional fragments in addition to PTH₇₋₈₄ as comprising the total PTH.Prior PTH totals generally provided lower numbers than PTH totalsincluding PTH fragments in addition to PTH₇₋₈₄. These totals were lowerbecause PTH fragments other than large N-terminal fragments were notrecognized. The present invention acknowledges this previous flaw in PTHtotals in addition to the enhanced predictive and therapeutic benefitsof ratios derived from measuring PTH fragments in addition tocirculating N-terminal fragments.

The presently contemplated PTH ratios are useful to provide cutoffvaluations to determine whether a subject suffers from adynamic bonedisease (ADN), mild hyperparathyroidism (mild HPT) or severehyperparathyroidism (severe HPT). Frequently, the present ratios areuseful for initial diagnosis. However, these ratios may be equallyuseful to monitor and guide therapy for subjects. In one preferredembodiment, the present ratios are utilized in conjunction withmeasurement of CAP and/or whole PTH levels. Table 1 below provides areference table for bone turnover rates as they relate to whole PTHlevels and PTH ratios consisting of PTH₁₋₈₄ versus PTH₇₋₈₄.

TABLE 1 Adynamic Bone Normal Turnover High Turnover Disease RatioBetween about Greater than about 2.0 Less than about PTH₁₋₈₄/ 1.4 toabout 2.0 1.4 PTH₇₋₈₄ PTH level Between about Greater than about 170Less than about PTH₁₋₈₄ 90-170 90 (pg/ml)

While not bound by theory, in light of current standards and practices,reliance solely on the values and ratios in Table 1 for determinationand differentiation between the three listed bone turnover ratecategories would be misplaced without reference to additional dataincluding, for example, bone histology information and/or additionalfragment related information (as provided herein).

Example ratios include variations of PTH parameters comprised of wholePTH (wPTH), C-terminal PTH (cPTH), and mid-terminus PTH fragments(mPTH). Depending on the antibodies utilized, cross-reactivity betweenthese parameters may be witnessed (and corrected). A non-limiting listof example ratios of the present disclosure include wPTH/cPTH,wPTH/mPTH, wPTH/(cPTH+mPTH), wPTH/(cPTH−wPTH)+(mPTH−wPTH),wPTH/(cPTH+mPTH−wPTH), wPTH/(wPTH+mPTH+cPTH), cPTH/mPTH, mPTH/cPTH,cPTH/wPTH, mPTH/wPTH, and so forth. One of skill in the art wouldrecognize that the inverse of these PTH ratios and other combinations ofthese parameters are equally suitable in the present methods. Moreover,CAP may be utilized as a PTH ratio parameter and used in conjunctionwith whole PTH levels, total PTH levels and/or PTH fragment levels inthe contemplated ratios.

For example, Table 2 below provides a series of PTH ratio cutoff values.Antibodies specific for whole PTH (wPTH), mid-terminus PTH fragments(mPTH), and C-terminal PTH (cPTH) fragments and corresponding histologydata are utilized to generate PTH level raw data providing the basevalues for ratio cutoffs. Each of the ratios presented below aregenerated based on the levels of cPTH, mPTH and/or wPTH as depicted.

TABLE 2 Cutoff Ratio composition (about) Indication wPTH/(cPTH + mPTH)<0.020 ADN wPTH/(cPTH + mPTH) >0.020 Severe HPT wPTH/(cPTH + mPTH) 0.020Mild HPT wPTH/((cPTH − wPTH) + (mPTH − wPTH)) <0.0185 ADN wPTH/((cPTH −wPTH) + (mPTH − wPTH)) >0.0185 Severe HPT wPTH/((cPTH − wPTH) + (mPTH −wPTH)) >0.0185 Mild HPT wPTH/(cPTH + mPTH − wPTH) <0.020 ADNwPTH/(cPTH + mPTH − wPTH) >0.020 Severe HPT wPTH/(cPTH + mPTH − wPTH)0.020 Mild HPT wPTH/(wPTH + mPTH + cPTH) <0.0175 ADN wPTH/(wPTH + mPTH +cPTH) >0.0175 Severe HPT wPTH/(wPTH + mPTH + cPTH) >0.0175 Mild HPTwPTH/cPTH <0.103 ADN wPTH/cPTH >0.103 Severe HPT wPTH/cPTH 0.103 MildHPT wPTH/mPTH <0.0225 ADN wPTH/mPTH >0.0225 Severe HPT wPTH/mPTH >0.0225Mild HPT

While Table 2 presents cutoff ranges for each of three clinicalindications, variation for each may exist. The term “about” is used witheach cutoff as a particularly preferred value range having slightinherent variation which may include the cutoff point as well. One ofskill in the art would understand that PTH assay parameters could bechanged such that the cutoff point may vary from that provided above;such variation falls within the scope of the present disclosure. In oneembodiment, the cutoff point represents a median cutoff value for anindication. In a preferred embodiment, the cutoff point represents arange below or above which a majority or all subjects having aparticular indication fall within.

There is a tremendous need to be able to non invasively separate thedialysis patients with ADN from those suffering from high bone turnoverto avoid over treatment of ADN dialysis patients. Over treatment ofdialysis patients with ADN is a frequent occurrence under presentlyutilized methods. For example, calcium based phosphate binders such asZemplar® and Calcijex® (Abbott Laboratories), for example, have beenused to treat dialysis patients. Under the treatment protocols utilizedand recommended, these patients are at a great risk of over treatmentdue to inaccurate measurement of PTH levels (including wPTH and PTHfragment levels). For example, the proportion of dialysis patientstreated with calcium based phosphate binders that become afflicted withADN rose sharply during the time spanning 1995 to 2000 from 12% to 48%of such patients. See, e.g., Malluche, H. H., The Importance of BoneHealth in ESRD: Out of the Frying Pan, Into the Fire?, World Congress onNephrology, Berlin, Germany (June 2003) (based on unpublished data). Itis postulated that this increase is due, in large part, to overtreatment of dialysis patients; and this over treatment of dialysispatients is due, in turn, to ineffective PTH level monitoring (includingwhole PTH and PTH fragment levels). Moreover, K/DOQI recommends wholePTH as the only marker useful for separating ADN from HBT dialysispatients. See K/DOQI Clinical Practice Guidelines for Bone Metabolismand Disease in Chronic Kidney Disease, Draft Guideline Statements andTreatment Algorithms (February 2003). However, whole PTH levels fail toconsistently separate ADN from HBT dialysis patients. See Qi, Q, et al.,Am. J. Kidney Dis. 26:622-31 (1995); Quarles, L D, et al., J. Clin.Endocrinol. Metab. 75:145-150 (1992). It is recognized herein that suchguidelines would appear to propagate the over treatment phenomenon.Accordingly, the present compositions and methods illustrate theeffectiveness of separating ADN from HBT dialysis patients via PTH ratioresults, rather than via measurement of whole PTH levels alone, andtheir use for routine clinical management of renal disease,osteoporosis, and/or dialysis patients.

D. PTH Assay Locations

The presently contemplated methods may be performed in a variety ofsettings and by a variety of entities. However, in general, the presentmethods and materials may be made available in a health care setting.Frequently, the present methods, e.g., determining and monitoring of PTHlevels and ratios as described herein, are utilized in the clinicalmanagement of disease or disorders in a subject by a care provider orclinical laboratory. A health care setting, as used herein, includesclinical laboratories, doctor's offices, hospitals, health managementorganization facilities, and outpatient care facilities, amongst avariety of other nontraditional settings useful for the delivery of careand subject testing.

E. PTH Sequences

The present disclosure contemplates the use of parathyroid hormonepeptides, peptide fragments, polynucleotides encoding whole PTH or PTHfragment peptides, and antibodies that specifically bind whole PTHand/or PTH fragments derived from a variety of mammalian sources. See,e.g., Caetano, A. R., et al., Equus Genome Res. 9(12): 1239-1249 (1999)(horse), U.S. Patent Application Publication US 2002/0110871 A1 (rat,mouse, bovine, canine, porcine), U.S. patent application Ser. Nos.09/344,639 and 09/231,422 (human). By way of nonlimiting example, PTHderived from the following sources and having the following peptidesequences are contemplated herein:

Human PTH₁₋₈₄ (SEQ ID NO: 1):SER-VAL-SER-GLU-ILE-GLN-LEU-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU-ASN-SER-MET-GLU-ARG-VAL-GLU-TRP-LEU-ARG-LYS-LYS-LEU-GLN-ASP-VAL-HIS-ASN-PHE-VAL-ALA-LEU-GLY-ALA-PRO-LEU-ALA-PRO-ARG-ASP-ALA-GLY-SER-GLN-ARG-PRO-ARG-LYS-LYS-GLU-ASP-ASN-VAL-LEU-VAL-GLU-SER-HIS-GLU-LYS-SER-LEU-GLY-GLU-ALA-ASP-LYS-ALA-ASP-VAL-ASN-VAL-LEU-THR-LYS-ALA-LYS-SER-GLN. Rat PTH₁₋₈₄ (SEQ ID NO: 2):ALA-VAL-SER-GLU-ILE-GLN-LEU-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU-ALA-SER-VAL-GLU-ARG-MET-GLN-TRP-LEU-ARG-LYS-LYS-LEU-GLN-ASP-VAL-HIS-ASN-PHE-VAL-SER-LEU-GLY-VAL-GLN-MET-ALA-ALA-ARG-GLU-GLY-SER-TYR-GLN-ARG-PRO-THR-LYS-LYS-GLU-ASP-ASN-VAL-LEU-VAL-ASP-GLY-ASN-SER-LYS-SER-LEU-GLY-GLU-GLY-ASP-LYS-ALA-ASP-VAL-ASP-VAL-LEU-VAL-LYS-ALA-LYS-SER-GLN. Mouse PTH₁₋₈₄ (SEQ ID NO: 3):ALA-VAL-SER-GLU-ILE-GLN-LEU-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU-ALA-SER-VAL-GLU-ARG-MET-GLN-TRP-LEU-ARG-ARG-LYS-LEU-GLN-ASP-MET-HIS-ASN-PHE-VAL-SER-LEU-GLY-VAL-GLN-MET-ALA-ALA-ARG-ASP-GLY-SER-HIS-GLN-LYS-PRO-THR-LYS-LYS-GLU-GLU-ASN-VAL-LEU-VAL-ASP-GLY-ASN-PRO-LYS-SER-LEU-GLY-GLU-GLY-ASP-LYS-ALA-ASP-VAL-ASP-VAL-LEU-VAL-LYS-SER-LYS-SER-GLN. Bovine PTH₁₋₈₄ (SEQ ID NO: 4):ALA-VAL-SER-GLU-ILE-GLN-PHE-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU-SER-SER-MET-GLU-ARG-VAL-GLU-TRP-LEU-ARG-LYS-LYS-LEU-GLN-ASP-VAL-HIS-ASN-PHE-VAL-ALA-LEU-GLY-ALA-SER-ILE-ALA-TYR-ARG-ASP-GLY-SER-SER-GLN-ARG-PRO-ARG-LYS-LYS-GLU-ASP-ASN-VAL-LEU-VAL-GLU-SER-HIS-GLN-LYS-SER-LEU-GLY-GLU-ALA-ASP-LYS-ALA-ASP-VAL-ASP-VAL-LEU-ILE-LYS-ALA-LYS-PRO-GLN. Canine PTH₁₋₈₄ (SEQ ID NO: 5):SER-VAL-SER-GLU-ILE-GLN-PHE-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU-SER-SER-MET-GLU-ARG-VAL-GLU-TRP-LEU-ARG-LYS-LYS-LEU-GLN-ASP-VAL-HIS-ASN-PHE-VAL-ALA-LEU-GLY-ALA-PRO-ILE-ALA-HIS-ARG-ASP-GLY-SER-SER-GLN-ARG-PRO-LEU-LYS-LYS-GLU-ASP-ASN-VAL-LEU-VAL-GLU-SER-TYR-GLN-LYS-SER-LEU-GLY-GLU-ALA-ASP-LYS-ALA-ASP-VAL-ASP-VAL-LEU-THR-LYS-ALA-LYS-SER-GLN. Porcine PTH₁₋₈₄ (SEQ ID NO: 6):SER-VAL-SER-GLU-ILE-GLN-PHE-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU-SER-SER-LEU-GLU-ARG-VAL-GLU-TRP-LEU-ARG-LYS-LYS-LEU-GLN-ASP-VAL-HIS-ASN-PHE-VAL-ALA-LEU-GLY-ALA-SER-ILE-VAL-HIS-ARG-ASP-GLY-GLY-SER-GLN-ARG-PRO-ARG-LYS-LYS-GLU-ASP-ASN-VAL-LEU-VAL-GLU-SER-HIS-GLN-LYS-SER-LEU-GLY-GLU-ALA-ASP-LYS-ALA-ALA-VAL-ASP-VAL-LEU-ILE-LYS-ALA-LYS-PRO-GLN. Horse PTH₁₋₈₆ (SEQ ID NO: 7):LYS-ARG-SER-VAL-SER-GLU-ILE-GLN-LEU-MET-HIS-ASN-LEU-GLY-LYS-HIS-LEU-ASN-SER-VAL-GLU-ARG-VAL-GLU-TRP-LEU-ARG-LYS-LYS-LEU-GLN-ASP-VAL-HIS-ASN-PHE-ILE-ALA-LEU-GLY-ALA-PRO-ILE-PHE-HIS-ARG-ASP-GLY-GLY-SER-GLN-ARG-PRO-ARG-LYS-LYS-GLU-ASP-ASN-VAL-LEU-ILE-GLU-SER-HIS-GLN-XXX-SER-LEU-GLY-GLU-ALA-ASP-LYS-ALA-ASP-VAL-ASP-VAL-LEU-SER-LYS-THR-LYS-SER-GLN.

VII. EXEMPLARY MODES FOR CARRYING OUT THE INVENTION

In disclosing the present invention, one should remember that there area number of closely analogous, species dependent forms of PTH (seeabove). The amino acid sequence of hPTH is shown in FIG. 1. However, forrat PTH, bovine PTH, or porcine PTH, for example, one finds thesubstitutions at some of the amino acids in the hPTH sequence (see,e.g., SEQ ID NOs: 1-7). For the purposes of the present invention, onecan use interchangeably antibodies or antibody fragments to forms ofthese PTHs, although it is preferred to use an antibody with specificityfor PTH having a sequence matching the species in which the PTHmeasurements are made.

A. Whole PTH Immunoassay

A preferred embodiment of the present invention is an immunoradiometricassay 15 (IRMA), often referred to as a sandwich assay, as shown FIGS. 2and 3. Elements employed in such an assay (10) include a captureantibody (12) attached to a solid support (14) and a signal antibody(16) having a label (18), attached thereto (20). Typically, one selectsa capture antibody that is specific for C-terminal PTH fragments (22),while the label antibody is specific for the initial wPTH peptidesequence which comprises a domain for adenylate cyclase activation (24),as shown in FIG. 2. However, one could reverse the specificity of theseantibodies, as is shown in FIG. 3.

Alternatively, one could create an immunoassay in which wPTH is eitherprecipitated from solution or otherwise differentiated in a solution, asin conventional precipitating assays or turbidometric assays. Forexample, one can use at least three antibodies to form a precipitatingmass. In addition to the initial wPTH sequence antibody and a C-terminalantibody, one can use at least a third antibody which attaches to themid portion of Pm. The combined mass of wPTH and the at least threeantibodies would form a labeled precipitating mass which can be measuredby conventional techniques.

Another method would be to couple the initial wPTH sequence antibody tocolloidal solid supports, such as latex particles. More specifically,one can create a signal antibody by iodinating 50 micrograms of affinitypurified goat anti-(1-6) PTH antibody (Scantibodies Laboratory, Inc.,Santee Calif., U.S.A.) by oxidation with chloramine T, incubation for 25seconds at room temperature with 1 millicurie of 125-1 radioisotope andreduction with sodium metabisulfate. Unincorporated 125-1 radioisotopeis separated from the 125-1-Goat anti-(1-6) PTH signal antibody by,passing the iodination mixture over a PD-10 desalting column (Pharmacia,Uppsala, Sweden) and following the manufacturers instructions. Thefractions collected from the desalting column are measured in a gammacounter and those fractions representing the 125-1-goat anti-(1-6) PTHantibody are pooled and diluted to approximately 300,000 DPM(disintegrations per minute) per 100 microliters. This solution is thetracer solution to be used in the whole PTH IRMA.

Capture antibody coated tubes can be created by attaching affinitypurified goat anti PTH 39-84 antibody, (Scantibodies Laboratory, Inc.,Santee, Calif., U.S.A.), to 12×75 mm polystyrene tubes (Nunc, Denmark)by means of passive absorption techniques which are known to those ofskill in the art. The tubes are emptied and dried, 20 creating solidphase antibody coated tubes.

To conduct a whole PTH assay of a sample, 200 microliter samples ofhuman serum are added to the solid phase antibody coated tubes. To eachtube is added 100 microliters of the tracer solution (labeled goatanti-(1-6) PTH signal antibody). The tubes 25 are incubated at roomtemperature with shaking at 170 rpm for 20-22 hours. During this timethe immunochemical reaction of forming the sandwich of {solid phase goatanti-(39-84) PTH antibody}—{ whole PTH}—{ 125-1-goat anti-(1-6) PTHantibody} takes place. Following this incubation, the test tubes arewashed with distilled water. Radioactivity on the solid phase, whichamount corresponds to the quantity of wPTH present, is measured using agamma counter. The radioactivity data for the samples is processed byconventional analysis with use of the results from standards andcontrols and computer software in order that the concentration of wholePTH in the samples may be ascertained. FIG. 4 shows a standard curve forsuch an assay.

B. Initial Whole PTH Sequence Peptide

In order to make the signal antibody in the above assays, first onemakes a synthetic PTH peptide corresponding either to human PTH₁₋₈, ratPTH₁₋₈, mouse PTH₁₋₈, bovine PTH₁₋₈, canine PTH₁₋₈, porcine PTH₁₋₈,horse PTH₁₋₈, human PTH₁₋₁₅, rat PTH₁₋₁₅, mouse PTH₁₋₁₅, bovine PTH₁₋₁₅,canine PTH₁₋₁₅, porcine PTH₁₋₁₅, or horse PTH₁₋₁₅, or at least fouramino acids in the common sequence. Although not bound by theory,suitable synthetic PTH peptides may extend beyond the PTH₁₋₈ asillustrated above, i.e., to position 84 of PTH₁₋₈₄. For example, any ofthe variety of PTH N-terminal fragments are suitable initial peptidesequences for this aspect. The selected peptide can play two roles inmaking an assay, first as a specific source for creating a polyclonalantibody or monoclonal antibody source for signal antibody or captureantibody, and second as part of an affinity purification means forisolating the desired signal antibody or capture antibody.

Briefly, such a peptide can be synthesized on an Applied Biosystems,Inc. (Foster City, Calif., U.S.A.) Model 431 automated peptidesynthesizer employing Fmoc (9-fluoronylmethoxycarbonyl) as thealpha-amino protecting group. All amino acids and solvents are fromApplied Biosystems and are of synthesis grade. Following synthesis, thepeptide is cleaved from the resin, and side chains are de-blocked, usinga cleavage cocktail 20 containing 6.67% phenol, 4.4% (v/v) thioanisoleand 8.8% ethanedithiol in trifluoroacetic acid (TFA). The cleavedpeptide is precipitated and washed several times in cold diethyl ether.It is then dissolved in water and lyophilized. The crude peptide issubjected to amino acid analysis (Waters PICO-TAG System, Boston, Mass.,U.S.A.) and reversed-phase HPLC using a VYDAC (TM) C8 column with 0.1%TFA in water and 25 99.9% acetonitrile in 0.1% TF A as the mobilebuffers. The presence of a single major peak along with the appropriateamino acid composition is taken as evidence that the peptide is suitablefor further use.

The resulting peptide is then attached to cross linked agarose beads(activated Sepharose 4B from Pharmacia, Uppsala, Sweden) according toinstructions from the manufacturer. Armed with the initial peptidesequence on a bead, one can affinity purify a polyclonal antibody serumsource to isolate the initial sequence antibody for the wPTHimmunoassay.

In a particularly preferred embodiment, initial sequence PTH antibodiessuccessfully distinguish between initial PTH peptides and C-terminal andmid-terminal PTH peptides, such that they specifically bind initialsequence PTH peptides.

In another preferred embodiment, the above methods are utilized tosynthesize mid-terminal and C-terminal PTH peptide fragments. Aftersynthesis of these peptides, the above methods are utilized to generateand isolate mid-terminal PTH antibodies and/or C-terminal PTHantibodies. In a preferred embodiment, mid-terminal PTH antibodiesspecifically bind mid-terminal PTH peptide fragments. In anotherpreferred embodiment, C-terminal PTH antibodies specifically bindC-terminal PTH peptide fragments.

C. Initial Sequence Whole PTH Antibody

In another embodiment, to create an affinity-purified initial sequencewhole PTH antibody, one first uses a PTH sequence peptide having anintact N-terminal as described above as part of an immunogen forinjection into a goat. For example, a PTH peptide ranging from PTH₁₋₃₄to PTH₁₋₈₄ may be utilized. The peptide can be used either by itself asan injectible immunogen, incorporated into a non PTH peptide having amolecular weight, typically, of between about 5,000 and 10,000,000, oras part of the wPTH complete sequence. The immunogen is mixed with anequal volume of Freund's complete adjuvant which is a mixture of lightmineral oil, Arlacel detergent, and inactivated mycobacteriumtuberculosis bacilli. The resulting mixture is homogenized to produce anaqueous/oil emulsion which is injected into the animal (typically agoat) for the primary immunization. The immunogen dose is approximately50-400 micrograms. The goats are injected monthly with the same dose ofimmunogen complex except no mycobacterium tuberculosis bacilli is usedin these subsequent injections. The goats are bled monthly,approximately three months after the 20 primary immunization. The serum(or antiserum) is derived from each bleeding by separating the red bloodcells from the blood by centrifugation and removing the antiserum whichis rich in initial sequence PTH antibodies.

To purify the antiserum for the desired initial sequence whole PTHantibody, one packs a separation column with a PTH sequence peptide(e.g., a PTH peptide ranging from PTH₁₋₅ to PTH₁₋₁₅) bound beadsdescribed above, washes the column and equilibrates it with 0.01 Mphosphate buffered saline (PBS). The antiserum is loaded onto the columnand washed with 0.01 M PBS in order to remove antibodies without theinitial sequence PTH specificity. The bound specific goat initialsequence PTH polyclonal antibody is eluted from the solid phase thatincludes optionally PTH₁₋₅ to PTH₁₋₁₅ in the column by passing anelution solution of 0.1 M glycine hydrochloride buffer, pH 2.5 throughthe column. The eluted polyclonal antibody is neutralized after itleaves the column with either the addition of 1.0 M phosphate buffer, pH7.5 or by a buffer exchange with 0.01 M PBS, as is known to those ofskill in the art. The polyclonal antibody is stored at 2-8 degreescentigrade.

In another embodiment, to create an affinity-purified anti-(1-6) PTHantibody, one first uses a selected initial PTH sequence peptide asdescribed above as part of an immunogen for injection into a goat. Thepeptide can be used either by itself as an injectible immunogen,incorporated into a non PTH peptide having a molecular weight,typically, of between about 5,000 and 10,000,000, or as part of the wPTHcomplete sequence. The immunogen is mixed with an equal volume ofFreund's complete adjuvant which is a mixture of light mineral oil,Arlacel detergent, and inactivated mycobacterium tuberculosis bacilli.The resulting mixture is homogenized to produce an aqueous/oil emulsionwhich is injected into the animal (typically a goat) for the primaryimmunization. The immunogen dose is approximately 50-400 micrograms. Thegoats are injected monthly with the same dose of immunogen complexexcept no mycobacterium tuberculosis bacilli is used in these subsequentinjections. The goats are bled monthly, approximately three months afterthe 20 primary immunization. The serum (or antiserum) is derived fromeach bleeding by separating the red blood cells from the blood bycentrifugation and removing the antiserum which is rich in (1-6) PTHantibodies.

To purify the antiserum for the desired (1-6) PTH antibody, one packs aseparation column with the initial PTH sequence peptide bound beadsdescribed above, washes the column and equilibrates it with 0.01 Mphosphate buffered saline (PBS). The antiserum is loaded onto the columnand washed with 0.01 M PBS in order to remove antibodies without the(1-6) PTH specificity. The bound specific goat anti-(1-6) PTH polyclonalantibody is eluted from the solid phase PTH 1-6 in the column by passingan elution solution of 0.1 M glycine hydrochloride buffer, pH 2.5through the column. The eluted polyclonal antibody is neutralized afterit leaves the column with either the addition of 1.0 M phosphate buffer,pH 7.5 or by a buffer exchange with 0. 01 M PBS, as is known to those ofskill in the art. The polyclonal antibody is stored at 2-8 degreescentigrade.

One of skill in the art would understand that there are acceptablevariations in the above practices. See, e.g., Harlow E, Lane D:Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988; Kohler & Milstein, Nature, 256: 495-7(1975). While not bound by theory, the above practices are suitable forproduction of other PTH N-terminal antibodies using selected N-terminalPTH sequence peptides as described herein.

In a particularly preferred embodiment, initial sequence PTH antibodiessuccessfully distinguish between initial PTH peptides and bothC-terminal and mid-terminus PTH peptides, such that they specificallybind initial sequence PTH peptides.

D. C-Terminal Sequence PTH Antibody

To create an affinity-purified anti-(60-84) PTH antibody, one first usesa selected C-terminal PTH sequence peptide as described above as part ofan immunogen for injection into a goat. In another embodiment, theimmunogen comprises whole PTH peptide, e.g., PTH₁₋₈₄. The peptide can beused either by itself as an injectible immunogen, incorporated into anon PTH peptide having a molecular weight, typically, of between about5,000 and 10,000,000. The immunogen is mixed with an equal volume ofFreunds complete adjuvant which is a mixture of light mineral oil,Arlacel detergent, and inactivated mycobacterium tuberculosis bacilli.The resulting mixture is homogenized to produce an aqueous/oil emulsionwhich is injected into the animal (typically a goat) for the primaryimmunization. The immunogen dose is approximately 50-400 micrograms. Thegoats are injected monthly with the same dose of immunogen complexexcept no mycobacterium tuberculosis bacilli is used in these subsequentinjections. The goats are bled monthly, approximately three months afterthe 20 primary immunization. The serum (or antiserum) is derived fromeach bleeding by separating the red blood cells from the blood bycentrifugation and removing the antiserum which contains (60-84) PTHantibodies. In another embodiment, the antiserum is removed whichcontains (60-84) PTH antibodies in addition to other PTH antibodies,e.g., whole PTH antibodies.

To purify the antiserum for the desired (60-84) PTH antibody, one packsa separation column with the C-terminal PTH sequence peptide bound beadsdescribed above, washes the column and equilibrates it with 0.01 Mphosphate buffered saline (PBS). The antiserum is loaded onto the columnand washed with 0.01 M PBS in order to remove antibodies without the(60-84) PTH specificity. The bound specific goat anti-(60-84) PTHpolyclonal antibody is eluted from the solid phase PTH 1-6 in the columnby passing an elution solution of 0.1 M glycine hydrochloride buffer, pH2.5 through the column. The eluted polyclonal antibody is neutralizedafter it leaves the column with either the addition of 1.0 M phosphatebuffer, pH 7.5 or by a buffer exchange with 0.01 M PBS, as is known tothose of skill in the art. The polyclonal antibody is stored at 2-8degrees centigrade.

One of skill in the art would understand that there are acceptablevariations in the above practices. See, e.g., Harlow E, Lane D:Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988; Kohler & Milstein, Nature, 256: 495-7(1975). While not bound by theory, the above practices are suitable forproduction of other PTH mid-terminal antibodies using selectedmid-terminal PTH sequence peptides as described herein. For example, PTHpeptides such as PTH₅₃₋₈₄, PTH₆₀₋₈₄, PTH₆₄₋₈₄, PTH₆₅₋₈₄, PTH₃₉₋₈₄,PTH₂₃₋₈₄, PTH₁₉₋₈₄, and other C-terminal PTH peptides as describedabove.

In a particularly preferred embodiment, C-terminal PTH antibodiessuccessfully distinguish between C-terminal peptides and both initialsequence and mid-terminus PTH peptides, such that they specifically bindC-terminal PTH peptides.

E. Mid-Terminus Sequence PTH Antibody

To create an affinity-purified anti-(44-68) PTH antibody, one first usesa selected mid-terminus PTH sequence peptide as described above as partof an immunogen for injection into a goat. In another embodiment, theimmunogen comprises whole PTH peptide, e.g., PTH₁₋₈₄. The peptide can beused either by itself as an injectible immunogen, incorporated into anon PTH peptide having a molecular weight, typically, of between about5,000 and 10,000,000. The immunogen is mixed with an equal volume ofFreunds complete adjuvant which is a mixture of light mineral oil,Arlacel detergent, and inactivated mycobacterium tuberculosis bacilli.The resulting mixture is homogenized to produce an aqueous/oil emulsionwhich is injected into the animal (typically a goat) for the primaryimmunization. The immunogen dose is approximately 50-400 micrograms. Thegoats are injected monthly with the same dose of immunogen complexexcept no mycobacterium tuberculosis bacilli is used in these subsequentinjections. The goats are bled monthly, approximately three months afterthe 20 primary immunization. The serum (or antiserum) is derived fromeach bleeding by separating the red blood cells from the blood bycentrifugation and removing the antiserum which contains (44-68) PTHantibodies. In another embodiment, the antiserum is removed whichcontains (44-68) PTH antibodies in addition to other PTH antibodies,e.g., whole PTH antibodies.

To purify the antiserum for the desired (44-68) PTH antibody, one packsa separation column with the mid-terminus PTH sequence peptide boundbeads described above, washes the column and equilibrates it with 0.01 Mphosphate buffered saline (PBS). The antiserum is loaded onto the columnand washed with 0.01 M PBS in order to remove antibodies without the(44-68) PTH specificity. The bound specific goat anti-(44-68) PTHpolyclonal antibody is eluted from the solid phase PTH 1-6 in the columnby passing an elution solution of 0.1 M glycine hydrochloride buffer, pH2.5 through the column. The eluted polyclonal antibody is neutralizedafter it leaves the column with either the addition of 1.0 M phosphatebuffer, pH 7.5 or by a buffer exchange with 0.01 M PBS, as is known tothose of skill in the art. The polyclonal antibody is stored at 2-8degrees centigrade.

One of skill in the art would understand that there are acceptablevariations in the above practices. See, e.g., Harlow E, Lane D:Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988; Kohler & Milstein, Nature, 256: 495-7(1975). While not bound by theory, the above practices are suitable forproduction of other PTH mid-terminus antibodies using selectedmid-terminus PTH sequence peptides as described herein. For example, PTHpeptides such as PTH₄₄₋₆₀, PTH₇₋₅₃, PTH₁₂₋₅₃, PTH₁₇₋₅₃, PTH₂₂₋₅₃,PTH₂₇₋₅₃, PTH₃₀₋₃₅, PTH₃₂₋₅₃, PTH₃₂₋₅₃, PTH₃₇₋₅₃, PTH₄₂₋₅₃, PTH₄₇₋₅₃,and other mid-terminal PTH peptides as described above.

In a particularly preferred embodiment, mid-terminus PTH antibodiessuccessfully distinguish between mid-terminus peptides and both initialsequence and C-terminal PTH peptides, such that they specifically bindmid-terminus PTH peptides.

F. Comparison Between Second Generation Whole PTH and Total PTH Assays

The present whole IRMA assay was compared to a conventional intact PTHor I-PTH immunoassay, the Allegro Nichols Intact-PTH assay, (which iscommercially available and made by Nichols Institute Diagnostics of SanJuan Capistrano, Calif., U.S.A.), in both PTH normal persons and thosesuffering from chronic uremia. This I-PTH immunoassay, detects bothPTH₇₋₈₄ and wPTH (see FIG. 10).

FIG. 5 shows the results for 34 normal human serum samples from healthysubjects which were assayed both by the present wPTH IRMA and the aboveI-PTH assay. In every case, the level of wpm detected by the IRMA islower than that reported by the I-PTH assay, demonstrating the abilityof the present IRMA to avoid detecting the interfering large, non (1-84)PTH fragment detected by the I-pm assay, (see FIG. 11). FIG. 6illustrates how such interference can occur. An N-terminal PTH specificsignal antibody which is not specific to the initial PTH peptidesequence, as in the present invention, can detect not only wPTH (as inthe upper part of FIG. 6), but also can detect PIN, the large, non(1-84) PTH fragment, (as in the lower part of FIG. 6).

A comparison of assay results for 157 chronic uremic patients is shownin FIG. 7. Serum samples from these patients were measured using thewPTH IRMA and the above I-PTH assay. In every case the wPTH levels arelower than I-PTH values.

G. Cyclase Active PTH/PTH Fragment Ratio Assay

Another preferred embodiment of the present invention is animmunoradiometric assay (IRMA), often referred to as a sandwich assay.As described herein, elements employed in such an assay include acapture antibody attached to a solid support and a signal antibodyhaving a label, attached thereto. Typically, one selects a captureantibody that is specific for C-terminal PTH fragments, while the labelantibody is specific for a mid-terminus PTH peptide sequence asdescribed herein. However, one could reverse the specificity of theseantibodies.

Alternatively, one could create an immunoassay in which PTH fragmentsare either precipitated from solution or otherwise differentiated in asolution, as in conventional precipitating assays or turbidometricassays. For example, one can use at least two or more antibodies to forma precipitating mass. These antibodies having specificity forN-terminal, C-terminal and/or mid-terminal portions of PTH. The combinedmass of the PTH fragment and the antibodies would form a labeledprecipitating mass which can be measured by conventional techniques.

Another method would be to couple C-terminal PTH sequence antibody tocolloidal solid supports, such as latex particles. In addition, a signalantibody can be created by iodinating 50 micrograms of affinity purifiedgoat anti-(7-53) PTH antibody by oxidation with chloramine T, incubationfor 25 seconds at room temperature with 1 millicurie of 125-1radioisotope and reduction with sodium metabisulfate. Unincorporated125-1 radioisotope is separated from the 125-1-Goat anti-(7-53) PTHsignal antibody by, passing the iodination mixture over a PD-10desalting column (Pharmacia, Uppsala, Sweden) and following themanufacturers instructions. The fractions collected from the desaltingcolumn are measured in a gamma counter and those fractions representingthe 125-1-goat anti-(7-53) PTH antibody are pooled and diluted toapproximately 300,000 DPM (disintegrations per minute) per 100microliters. This solution is the tracer solution to be used in the PTHassay.

Other signal antibodies can also be provided using affinity purifiedgoat anti-(12-53) PTH antibody, purified goat anti-(17-53) PTH antibody,purified goat anti-(22-53) PTH antibody, purified goat anti-(27-53) PTHantibody, purified goat anti-(32-53) PTH antibody, purified goatanti-(37-53) PTH antibody, purified goat anti-(42-53) PTH antibody,purified goat anti-(47-53) PTH antibody, and the like. Antibodiesspecific for other mid-terminus PTH peptide fragments and epitopesdescribed herein are also contemplated. Optimally, PTH antibodiesspecific for different PTH portions are obtained utilizing differentgoats and ELISA methods may be utilized to determine optimum antibodygeneration and use. See, e.g., Harlow E, Lane D: Antibodies: Alaboratory manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988.

Capture antibody coated tubes can be created by attaching affinitypurified goat anti-(39-84) PTH antibody to 12×75 mm polystyrene tubes(Nunc, Denmark) by means of passive absorption techniques which areknown to those of skill in the art. The tubes are emptied and dried, 20creating solid phase antibody coated tubes. The present methods are alsouseful to create solid phase antibody coated tubes having any of avariety of C-terminal PTH antibodies. For example, antibodies generatedspecific for PTH₄₀₋₈₄ ranging to those specific for PTH₆₀₋₈₄ andPTH₆₅₋₈₄ are useful in the present methods. One of skill in the artwould understand that the specificity for the capture and label/signalantibodies may be reversed.

A selection of these specific antibodies may be utilized to assaysamples from patients having adynamic low bone turnover. In one example,a sample of 50-100 ml blood is drawn from a subject having adynamic lowbone turnover. The sample is then subject to multiple sandwich assaysutilizing the capture and label antibodies described herein above. Eachassay determines the level of whole PTH, in addition to one or more PTHfragments present in the sample. The level of whole PTH is then comparedwith a variety of permutations of PTH fragment levels and total PTHlevels. The comparison results are then viewed in light of correspondingbone histological data for the patient to determine one or morecomparison schemes that are predictive of bone turnover rate. Ratioresults that are produced through the practice of this assay arerepresented in Table 1 and are further described hereinbefore.

A PTH ratio is predictive of certain bone turnover rates in a subject.The present disclosure recognizes that additional PTH fragments may bepresent in a sample beyond PTH₇₋₈₄, and often effect the bone turnoverrate. Thus, the present compositions, kits and methods are particularlyuseful to account for PTH fragments in addition to PTH₇₋₈₄, havingassociated therapeutic and predictive benefits for a subject. Althoughnot bound by theory, one component of this discovery is illustrated, inpart, by the observation that although a percentage of circulating largePTH fragments are inactive, others exhibit antagonistic effects to thoseexhibited by whole PTH. Thus, accounting for all, or a majority of, thecirculating PTH fragments in a particular assay will reduce thevariability of the result, and increase its therapeutic, diagnostic andpredictive potential.

H. Clinical Use

The present wPTH and PIN assays have been used in a clinical settinginvolving 188 persons. The group included 31 persons having normalhealthy parathyroid glands and 157 patients with chronic uremia who areundergoing dialysis on a continuous basis. Each person had a bloodsample drawn which was assayed using a wPTH assay from ScantibodiesLaboratory, Inc. as well as an I-PTH assay from Nichols Institute whichgave total PTH values.

Table 3 shows the results individually and comparatively, of the wPTH,PIN, and 10 total PTH assays from chronic uremic patients on dialysis.

TABLE 3 Patient Total PTH CAP CIP CIP to CIP to CAP to No. pg/ml pg/mlpg/ml Total PTH CAP Total PTH 1 1410 740 670 48% 91% 52% 2 185 89 96 52%108% 48% 3 231 104 127 55% 122% 45% 4 1020 590 430 42% 73% 53% 5 270 159111 41% 70% 59% 6 201 100 101 50% 101% 50% 7 380 100 280 74% 280% 26% 8460 277 183 40% 66% 60% 9 380 197 183 48% 93% 52% 10 880 522 358 41% 69%59% 11 310 154 156 50% 101% 50% 12 880 451 429 49% 95% 51% 13 670 418252 38% 60% 63% 14 390 221 169 43% 76% 57% 15 170 108 62 36% 57% 64% 16510 381 129 25% 34% 75% 17 200 67 133 67% 199% 34% 18 170 109 61 36% 56%64% 19 360 199 161 45% 81% 55% 20 260 164 96 37% 59% 63% 21 440 372 6815% 18% 85% 22 120 51.7 68.3 57% 132% 43% 23 600 527 73 12% 14% 83% 24220 130 90 41% 69% 59% 25 190 136 54 28% 40% 72% 26 220 118 102 46% 86%54% 27 630 334 296 47% 89% 53% 28 150 90 60 40% 67% 60% 29 170 106 6438% 60% 62% 30 810 489 321 40% 66% 60% 31 570 319 251 44% 79% 56% 32 570133 103 18% 22% 82% 33 400 564 100 25% 33% 75% 34 560 89 182 33% 48% 68%35 310 121 189 61% 156% 39% 36 240 98 142 59% 145% 41% 37 280 133 15754% 118% 48% 38 230 124 106 46% 85% 54% 39 350 319 31 9% 10% 91% 40 200133 67 34% 50% 67% 41 920 564 356 39% 63% 61% 42 210 89 121 58% 136% 42%43 1990 904 1086 55% 120% 45% 44 300 212 88 29% 42% 71% 45 260 132 12849% 97% 51% 46 140 72 68 49% 94% 51% 47 250 129 121 48% 94% 52% 48 13072 58 45% 81% 56% 49 1840 1000 840 46% 84% 54% 50 280 167 113 40% 68%60% 51 490 268 222 45% 83% 55% 52 150 77.1 72.9 49% 95% 51% 53 140 58.181.9 59% 141% 42% 54 210 92.7 117.3 56% 127% 44% 55 160 79 81 51% 103%49% 56 480 296 184 38% 62% 62% 57 480 281 199 41% 71% 59% 58 270 120 15056% 125% 44% 59 97 45 52 54% 116% 46% 60 330 154 176 53% 114% 47% 61 11056 54 49% 96% 51% 62 660 456 204 31% 45% 69% 633 300 137 163 54% 119%46% 64 240 145 95 40% 66% 60% 65 100 66.5 33.5 34% 50% 67% 66 410 416.36.3 2% 2% 102% 67 410 235.7 174.3 43% 74% 57% 68 45 14.4 30.6 68% 213%32% 69 200 102.3 97.7 49% 96% 51% 70 300 134 166 55% 124% 45% 71 320 202118 37% 58% 63% 72 440 254 186 42% 73% 58% 73 190 99.6 90.4 48% 91% 52%74 160 74.6 85.4 53% 114% 47% 75 600 429.8 170.2 28% 40% 72% 76 1140 632508 45% 80% 55% 77 40 211 229 52% 109% 48% 78 450 276 174 39% 63% 61% 79510 344 166 33% 48% 67% 80 190 62.8 127.2 67% 203% 33% 81 170 86 84 49%98% 51% 82 180 103.4 76.6 43% 74% 57% 83 78 22.7 55.3 71% 244% 29% 84230 117 113 49% 97% 51% 85 160 96 64 40% 67% 60% 86 220 89 131 60% 147%40% 87 470 321.5 148.5 32% 46% 68% 88 310 137 173 56% 126% 44% 89 20501127 923 45% 82% 55% 90 930 414 516 55% 125% 45% 91 180 65 115 64% 177%36% 92 560 238 322 58% 135% 43% 93 640 597 43 7% 7% 93% 94 590 382 20835% 54% 65% 95 270 103 167 62% 162% 38% 96 560 349 211 38% 60% 62% 97180 78 102 57% 131% 43% 98 790 429 361 46% 84% 54% 99 670 372 298 44%80% 56% 100 140 20.4 119.6 85% 586% 15% 101 190 117 73 38% 62% 62% 102190 108 82 43% 76% 57% 103 430 217 213 50% 98% 50% 104 560 439 121 22%28% 78% 105 500 357.7 142.3 28% 40% 72% 106 1560 777 783 50% 101% 50%107 62 24.3 37.7 61% 155% 39% 108 430 226 204 47% 90% 53% 109 160 67.292.8 58% 138% 42% 110 530 346 184 35% 53% 65% 111 260 142 118 45% 83%55% 112 580 163 41 72% 256% 28% 113 440 579 139 32% 24% 132% 114 500232.3 267.7 54% 115% 46% 115 160 60 100 63% 167% 38% 116 340 202 138 41%68% 59% 117 260 138 122 47% 88% 53% 118 260 119 141 54% 118% 46% 119 16084 76 48% 90% 53% 120 130 46 84 65% 183% 35% 121 190 104 86 45% 83% 55%122 420 334 86 20% 26% 80% 123 630 440 190 30% 43% 70% 124 75 26.4 48.665% 184% 35% 125 260 143 117 45% 82% 55% 126 640 409 231 36% 56% 64% 127130 66.7 63.3 49% 95% 51% 128 700 381 319 46% 84% 54% 129 560 376 18433% 49% 67% 130 240 107 133 55% 124% 45% 131 110 63 47 43% 75% 57% 132420 297 123 29% 41% 71% 133 580 229 351 61% 153% 39% 134 310 201.2 108.835% 54% 65% 135 160 97.9 62.1 39% 63% 61% 136 290 138.7 151.3 52% 109%48% 137 200 96.2 103.8 52% 108% 48% 138 770 662.7 107.3 14% 16% 86% 139290 130.7 159.3 55% 122% 45% 140 260 219 41 16% 19% 84% 141 350 211 13940% 66% 60% 142 730 463.5 266.5 37% 57% 63% 143 490 231 259 53% 112% 47%144 160 87 73 46% 84% 54% 145 380 222 158 42% 71% 58% 146 210 93.5 116.555% 125% 45% 147 630 383.4 246.6 39% 64% 61% 148 150 83.2 66.8 45% 80%55% 149 320 152.5 167.5 52% 110% 48% 150 900 467.6 432.4 48% 92% 52% 1511180 818.6 361.4 31% 44% 69% 152 120 38.4 81.6 68% 213% 32% 153 52301388 3842 73% 277% 27% 154 34 10.5 23.5 69% 224% 31% 155 1020 590.6429.4 42% 73% 58% 156 280 76.6 103.4 57% 135% 43% 157 120 51.1 68.9 57%135% 43% Median 300 154 127 46% 84% 54%

TABLE 4 shows the results, individually and comparatively, of the wPTH,PIN, assays from the normals.

TABLE 4 Total PTH CAP CIP CIP to CIP to CAP to Patient No. pg/ml pg/mlpg/ml Total PTH CAP Total PTH 1 17.13 3.32 13.81 81% 416% 19% 2 32.9210.49 22.43 68% 214% 32% 3 31.32 10.31 21.01 67% 204% 33% 4 41.84 12.7229.12 70% 229% 30% 5 33.03 10.09 22.94 69% 227% 31% 6 44.32 14.23 30.0968% 211% 32% 7 31.47 6.80 24.67 78% 363% 22% 8 20.82 10.03 10.79 52%108% 48% 9 34.64 15.95 18.69 54% 117% 46% 10 23.69 5.25 18.44 78% 351%22% 11 53.98 17.82 36.16 67% 203% 33% 12 52.71 18.83 33.88 64% 180% 36%13 26.92 5.63 21.29 79% 378% 21% 14 39.93 11.86 28.07 70% 237% 30% 11548.84 20.47 28.37 58% 139% 42% 16 29.56 13.68 15.88 54% 116% 46% 1736.19 14.69 21.50 59% 146% 41% 18 20.96 6.99 13.97 67% 200% 33% 19 59.2927.89 31.40 53% 113% 47% 20 45.57 18.23 27.34 60% 150% 40% 21 35.6418.72 16.92 47% 90% 53% 22 38.53 19.56 18.97 49% 97% 51% 23 21.71 9.3412.37 57% 132% 43% 24 32.42 13.51 18.91 58% 140% 42% 25 28.50 10.4118.09 63% 174% 37% 26 18.27 7.80 10.37 57% 133% 43% 27 39.96 17.29 22.6757% 131% 43% 28 34.08 15.24 18.84 55% 124% 45% 29 42.95 19.59 23.36 54%119% 46% 30 38.40 12.16 26.24 68% 216% 32% 31 47.57 18.45 29.12 61% 158%39% MEDIAN 34.64 13.51 21.50 61% 158% 39%

Clearly, the statistically significant differences in the medians ofthese two groups demonstrates that one can differentiate between the twoby using these assays alone or by comparing their respective values.

TABLE 5 Total CIP to CAP to PTH CAP CIP Total CIP to Total Sample pg/mlpg/ml pg/ml PTH CAP PTH Type Median Median Median Median Median MedianChronic 300 154 127 46%  84% 55% uremia (n = 157) Normal 34.64 13.5121.50 61% 158% 37% (n = 31) P-Value <0.0001 <0.0001 <0.0001 <0.0001<0.0001 <0.0001I. Characterization of the “Whole” PTH IRMA Assay

This new Whole PTH two-site assay (Scantibodies Laboratories, Santee,Calif., USA) first employees and antibody that recognizes the 39-84region of the PTH molecule. This antibody, produced in a goat andaffinity purified, is present in relative excess and is immobilized ontopolystyrene-coated tubes. The second antibody, also developed in a goat,was also affinity purified and recognizes only the first six amino acids(1 to 6; Ser-Val-Ser-Glu-Ile-Gln) of the human PTH molecule (FIG. 1).This anti-hPTH assay uses synthetic human PTH 1-84 as the standard, witha limit of detection of approximately 1 to 2 pg/mL. Normal values rangefrom 5 to 35 pg/mL. The interassay and intra-assay coefficients ofvariation were found to be between 2 and 7% and recovery was from 96 to106%. The Whole PTH assay was compared with the Intact PTH assaypurchases from the Nichols Institute (I-Nichols, San Jan Capistrano,Calif., USA). Synthetic human PTH 1-84 and 7-84 were purchased fromBachem (Torrance, Calif. USA). To assess circulating levels of hPTH 1-84and non-(1-84) PTH, heparinized blood samples were obtained beforedialysis from 28 patients who had been maintained on chronichemodialysis for 1.2 to 7.5 years and from 14 renal transplant patients(1 to 6 years).

1. Studies in Vitro

Osteoblastic cell line. To compare the biological effects of the twopeptides (HPTH 1-84 and 7-84), intracellular cAMP production wasmeasured in the rate osteosarcoma cell line ROS/17.2, which has anosteoblastic phenotype and is known to increase cAMP production inresponse to PTH. Cells were cultured in Ham's F12 media containing 10%fetal bovine serum. Cells were plated out in 12-well plates at a densityof 30,000 cells per well and grown to confluence. Cells were washedthree times with KHMS buffer at 37° C. (KCl 4.0 mmol/L, CaCl₂ 1.25mmol/L, MgSO₄ 1.25 mmol/L, KH₂PO₄ 1.2 mmol/L, HEPES 10 mmol/L, NaCl 100mmol/L, NaHCO₃ 37 mmol/L, and glucose 10 mmol/L, pH 7.5). cAMPproduction was measured using 500 μL of KHMS buffer (37°) containingisobutyl-1 methylxantine (IBMX) 1.0 mmol/L and various concentrations(10⁻¹¹ to 10⁻⁸ mol/L) of hPTH 1-84 or hPTH 7-84. After a five-minuteincubation, 100 μL of 1.8 mol/L pechloric acid were added. After anadditional five-minute incubation at room temperature, 100 μL of 3mol/KHCO₃ were added to neutralize the acid. Samples were centrifuged at3000 rpm for 15 minutes, and the supernatants were assayed for cAMP[26].

Analysis of PTH in human parathyroid glands. Human parathyroid glandswere placed in ice-cold phosphate-buffered saline and processed within30 minutes of parathyroidectomy. Aliquots of parathyroid tissue weredissected, weighed, and homogenized in 500 μL of a buffer containing 100mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 1 mol/L DL-dithiothreitol, anda complete TM protease inhibitor cocktail (Boehringer-Mannheim,Mannheim, Germany). Homogenates were sonicated three times for 30seconds each at 0° C. and centrifuged at 10,000× g for 15 minutes.Supernatants were kept at −70° C. until measurements of 1-84 PTH,non-(1-84) PTH, and total protein were performed.

2. Studies in Vivo

Calcemic response. Normal female Sprague-Dawley rats weighing 225 to 250g (Harlan, Indianapolis, Ind., USA) were parathyroidectomized (PTX) andfed a 0.02% calcium diet. Rats with a plasma calcium below 7.0 mg/dLafter overnight fasting were included in the study. A 20 μg dose of hPTH1-84 or 7-84 was given intraperitoneally to PTX rats in four doses of 5μg each at 30-minute intervals (0, 30, 60, and 90 minutes). For controlstudies, the rats received vehicle (saline solution) alone. Blood wasdrawn via the tail at 0, 60, 90, and 120 minutes. For competitionexperiments, rats received an injection of hPTH 7-84 10 minutes prior toeach injection of hPTH 1-84. The molar ratio of hPTH 7-84/hPTH 1-84 was1:1.

Phosphaturic response. Normal female Sprague-Dawley rats weighing 225 to250 g were prepared for clearance studies under light anesthesia.Polyethylene catheters (PE50) were placed in the femoeral artery for thecollection of blood and measurement of blood pressure (Blood PressureAnalyzer; Micro-Medic, Inc., Louisville, Ky., USA), in the femoral veinfor infusion and in the bladder for the collection of urine. Rats wereplaced in Plexiglase® holders and allowed to recover from the effect ofthe anesthetic for one hour. A priming dose (0.6 mL) of chemical inulinin saline was administered over a period of three minutes to achieve aplasma inulin level between 50 and 100 mg/mL. A solution of salinecontaining inulin to maintain this level and calcium gluconate todeliver 0.5 mg. calcium was infused at the rate of 0.03 mL/min. Afterequilibration, a total of four 30-minute urine collections was obtained.

To assess the effect of hPTH 1-84 on phosphate excretion, urine wascollected during two control periods, after which rats received apriming bolus of 1.8 μg of hPTH 1-84 followed by a sustained infusionthat delivered a total of 8.2 μg of I-PTH. After an equilibration periodof 20 minutes, two 30-minute urine collections were obtained. Incompetition experiments, hPTH 7-84 was given five minutes prior to hPTH1-84 at a molar ratio of 4:1.

Blood samples and blood pressure measurements were recorded at thebeginning and end of the baseline period, at the beginning of the PTHinfusion period, and at the end of the study. The concentration ofinulin in plasma and urine was determined by the method of Führ,Kaczmarczyk, and Kruttgen, Klin Wochenschr, 33:729-730 (1955). Theestimation of the glomerular filtration rate (GFR) by inulin clearanceand the calculation of the fractional urinary excretion rate ofphosphorus (FE_(P04)) were performed in the standard fashion. Bloodsamples were centrifuged, and plasma phosphorus and calciumconcentrations were measured.

3. Serum Chemistries

Total plasma calcium levels were determined using an atomic absorptionspectrophotometer (model 1100B; Perkin Elmer, Norwalk, Conn., USA).Plasma phosphorus levels were determined using an autoanalyzer (COBASMIRA Plus; Roche, Newark, N.J., USA).

4. Statistical Analysis

Results are expressed as mean±SEM. N indicates the number of samples.The paired t-test was employed to examine statistical significance,unless otherwise indicated in the text.

J. Specificity of IRMA Assays for hPTH 1-84

Initial studies compared the ability of the Nichols Intact (I-Nichols)PTH assay and the new Whole PTH assay to discriminate between the hPTH1-84 and hPTH 7-84 molecules. FIG. 13 shows that the Nichols “intact”PTH assay did not discriminate between human PTH 1-84 and 7-84. However,as depicted in FIG. 14, studies performed using the Whole PTH assay showthat hPTH 1-84 was detected with a high degree of sensitivity, whereashPTH 7-84 was undetectable, even at a concentration as high as 10,000pg/mL.

1. Studies in Vitro

The results of cAMP production by ROS/17.2 cells exposed to hPTH 1-84 orhPTH 7-84 are shown in FIG. 15. Unlike hPTH 7-84, hPTH 1-84 increasedcAMP production in a does-dependent manner. hPTH 1-84 (10⁻⁸ mol/L)increased intracellular cAMP from 18.1±1.25 to 738±4.13 nmol/well. Onthe other hand, the same concentration of hPTH 7-84 had no effect oncAMP (N=6).

2. Studies in Vivo in Rats

We next examined the hPTH 7-84 fragment as a potential competitiveinhibitor of hPTH 1-84 in bone by measuring changes in serum calcium inPTX rats. FIG. 12 shows that the administration of hPTH 1-84 to PTX ratsfed a 0.02% calcium diet increased plasma calcium by 0.65±0.10 mg/dL(N=9, P.<0.001, ANOVA). With the administration of vehicle alone, plasmacalcium changed slightly in accordance with PTX (−0.17±0.10 mg/dL, N=5).A slight but significant decrease was observed in the rats receivinghPTH 7-84 (0.30±0.08 mg/dL, N=5, P<0.05). When both peptides were giventogether in a 1:1 molar ratio, the calcemic response induced by theadministration of hPTH 1-84 alone decreased by 94% (N=6, P<0.001, ANOVA.Thus in this model, hPTH 7-84 significantly inhibits hPTH 1-84 inductionof bone calcium mobilization.

The phosphaturic effects of these two peptides were then evaluated (FIG.16). The GFR did not change in rats infused with hPTH 1-84 (1.8±0.3 vs.1.8±0.1 mL/min), whereas fractional excretion of phosphate (FE_(P04))increased from 11.9±2.4 to 27.7±2.4% (N=10, P<0.001). When hPTH 7-84 wasgiven simultaneously with hPTH 1-84, the GFR increased from 2.1±0.1 to2.6±0.2 mL/min (N=8, P<0.05). However, despite this increase in GFR, theincrease in FE_(P04) induced by treatment with hPTH 1-84 wassignificantly decreased by 50.2% (P<0.01). by virtue of theco-administration of hPTH 7-84.

3. Studies in Humans

FIG. 17 shows that the values for plasma PTH were higher in all 28patients on chronic dialysis when measured with the I-Nichols assaycompared with the Whole assay. The median PTH values were 523 versus 344pg/ml (P<0.001), respectively. A regression analysis of these data isshown in FIG. 7.

The association between plasma levels of non-(1-84) PTH, “likely” HPTH7-84, and plasma calcium and phosphorus was next examined in 20 patientsmaintained on chronic dialysis (FIG. 18). There was a positivecorrelation between the percentage of non-(1-84) PTH and serum calcium(P<0.002), but no correlation with plasma phosphorus (data not shown).These studies were performed only in those patients in whom there werevalues for calcium, phosphorous, and PTH from the same blood sample[20].

In a group of 14 renal transplant patients the percentage of non-(1-84)PTH was found to be 44.1±3.1% of the total PTH, as measured by theI-Nichols assay and the Whole PTH assay (FIG. 19). The absolute PTHvalue with the I-Nichols assay was 132.9±39.9 compared with 79.8±24.8pg/mL (P<0.005) with the Whole PTH assay.

Finally, we examined whether intracellular cleavage of the hPTH 1-84molecule occurs in the parathyroid gland, thus producing the non (1-84)PTH fragment. Surgically excised parathyroid glands from six uremicpatients maintained on chronic dialysis were studied. FIG. 20 shows thatnon (1-84) PTH fragments exist in the cell lysates from theseparathyroid glands and represent 41.8±3.2% (P<0.05) of the totalintracellular PTH measured by the “intact” PTH assay (that is, 1-84 PTHand most likely 7-84 PTH).

The ordinarily skilled artisan can appreciate that the present inventioncan incorporate any number of the preferred features described above.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims.

Citation of the above publications or documents is not intended as anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents.

1. An isolated antibody that specifically binds to an N-terminalsequence of whole parathyroid hormone (PTH) and is capable of detectingsaid whole PTH at a physiological level in a mammalian sample, with aproviso that said isolated antibody avoids binding to a non-whole PTHfragment.
 2. The isolated antibody of claim 1, which is a monoclonalantibody that specifically binds to whole PTH.
 3. The isolated antibodyof claim 1, which specifically binds to an epitope comprised in PTH₁₋₅.4. The isolated antibody of claim 1, which specifically binds to theparathyroid hormone peptide human PTH₁₋₈, rat PTH₁₋₈, mouse PTH₁₋₈,bovine PTH₁₋₈, canine PTH₁₋₈, porcine PTH₁₋₈, horse PTH₁₋₈, humanPTH₁₋₁₅, rat PTH₁₋₁₅, mouse PTH₁₋₁₅, bovine PTH₁₋₁₅, canine PTH₁₋₁₅,porcine PTH₁₋₁₅, or horse PTH₁₋₁₅, wherein at least four amino acids insaid peptide sequence are part of a reactive portion with the antibody.5. The isolated antibody of claim 1, which specifically binds to anepitope comprised in PTH₂₋₅, PTH₂₋₆, PTH₂₋₇, PTH₂₋₈, PTH₂₋₉, PTH₂₋₁₀,PTH₂₋₁₁, PTH₂₋₁₂, PTH₂₋₁₃, PTH₂₋₁₄, or PTH₂₋₁₅.
 6. The isolated antibodyof claim 1, wherein the binding between the antibody and the N-terminalsequence of whole PTH is dependent on the presence of amino acidresidues 2-5 of the hPTH.
 7. The isolated antibody of claim 1, whereinthe binding between the antibody and the N-terminal sequence of wholePTH is dependent on the presence of amino acid residues 3-6 of the hPTH.8. The isolated antibody or antibody fragment of claim 1, wherein thenon-whole PTH fragment is a peptide having an amino acid sequence frombetween PTH₃₋₈₄ and PTH₃₄₋₈₄.
 9. The isolated antibody of claim 1,wherein the non-whole PTH fragment is a peptide having an amino acidsequence of human PTH₇₋₈₄.
 10. A method for measuring a physiologicallevel of whole parathyroid hormone (PTH) in a mammalian sample, whichmethod comprises: a) obtaining a sample from a mammal to be tested; b)contacting said sample with an isolated antibody that specifically bindsto an N-terminal sequence of whole PTH and is capable of detecting saidwhole PTH at a physiological level in said mammalian sample, with aproviso that said isolated antibody avoids binding to a non-whole PTHfragment in said sample; and c) assessing a complex formed between saidwhole PTH, if present in said sample, and said antibody, to measurephysiological level of said whole PTH in said mammalian sample, whereinsaid isolated antibody specifically binds to an epitope comprised inPTH₁₋₅, PTH₁₋₆, PTH₁₋₇, PTH₁₋₈, PTH₁₋₉, PTH₁₋₁₀, PTH₁₋₁₁, PTH-₁₋₁₂,PTH₁₋₁₃, PTH₁₋₁₄ or PTH₁₋₁₅.
 11. The method of claim 10, wherein thesample is selected from the group consisting of a serum, a plasma and ablood sample.
 12. The method of claim 10, wherein the sample, is aclinical sample.
 13. The method of claim 10 which is used for clinicalmanagement of renal disease subjects, subjects afflicted withosteoporosis or diagnosing primary hyperparathyroidism.
 14. The methodof claim 10, wherein the mammal is a human.
 15. The method of claim 14,wherein the sample is a human clinical sample.
 16. The method of claim10, wherein the antibody is a monoclonal antibody that specificallybinds to whole PTH.
 17. The method of claim 10, wherein the antibodyspecifically binds to an epitope comprised in PTH₁₋₆, PTH₁₋₈, PTH₁₋₉,PTH₁₋₁₂, or PTH₁₋₁₅.
 18. The method of claim 10, wherein the antibodyspecifically binds to the PTH peptide human PTH₁₋₈, rat PTH₁₋₈, mousePTH₁₋₈, bovine PTH₁₋₈, canine PTH₁₋₈, porcine PTH₁₋₈, horse PTH₁₋₈,human PTH₁₋₁₅, rat PTH₁₋₁₅, mouse PTH₁₋₁₅, bovine PTH₁₅, canine PTH₁₋₁₅,porcine PTH₁₋₁₅, or horse PTH₁₋₁₅, wherein at least four amino acids insaid peptide sequence are part of a reactive portion with the antibody.19. The method of claim 10, wherein the non-whole PTH fragment isselected from the group consisting of PTH₃₋₈₄, PTH₄₋₈₄, PTH₅₋₈₄,PTH₆₋₈₄, PTH₇₋₈₄, PTH₈₋₈₄, PTH₉₋₈₄, PTH₁₀₋₈₄, PTH₁₁₋₈₄, PTH₁₂₋₈₄,PTH₁₃₋₈₄, PTH₁₄₋₈₄, PTH₁₅₋₈₄, PTH₁₆₋₈₄, PTH₁₇₋₈₄, PTH₁₈₋₈₄, PTH₁₉₋₈₄,PTH₂₀₋₈₄, PTH₂₁₋₈₄, PTH₂₂₋₈₄, PTH₂₃₋₈₄, PTH₂₄₋₈₄, PTH₂₅₋₈₄, PTH₂₆₋₈₄,PTH₂₇₋₈₄, PTH₂₈₋₈₄, PTH₂₉₋₈₄, PTH₃₀₋₈₄, PTH₃₁₋₈₄, PTH₃₂₋₈₄, PTH₃₃₋₄₄ andPTH₃₄₋₈₄.
 20. The method of claim 10, wherein the non-whole PTH fragmentis a peptide having an amino acid sequence of human PTH₇₋₈₄.
 21. Themethod of claim 10, wherein the complex is assessed by a sandwich assayformat.
 22. The method of claim 21, wherein the antibody thatspecifically binds to an N-terminal sequence of whole PTH is used as afirst antibody in a sandwich format assay, and a second antibody used inthe sandwich format assay is an antibody that is capable of binding to aportion of whole PTH other than the N-terminal sequence to which thefirst antibody binds.
 23. The method of claim 22, wherein either thefirst antibody or the second antibody is attached to a surface andfunctions as a capture antibody.
 24. The method of claim 23, wherein thecapture antibody is attached to the surface directly.
 25. The method ofclaim 23, wherein the capture antibody is attached to the surface via abiotin-avidin (or streptavidin) linking pair.
 26. The method of claim10, wherein the complex is assessed by a format selected from the groupconsisting of an enzyme-linked immunosorbent assay (ELISA),immunoblotting, immunoprecipitation, radioimmunoassay (RIA),immunostaining, latex agglutination, indirect hemagglutination assay(IHA), complement fixation, indirect immunofluorescent assay (IFA),nephelometry, flow cytometry assay, plasmon resonance assay,chemiluminescence assay, lateral flow immunoassay, u-capture assay,inhibition assay and avidity assay.
 27. The method of claim 10, whereinthe complex is assessed in a homogeneous assay format.
 28. The method ofclaim 10, wherein the physiological level of whole parathyroid hormonePTH is less than 4 pmol/L.
 29. The method of claim 10, wherein thephysiological level of whole PTH is from about 0.2 pmol/L to about 4pmol/L.
 30. The method of claim 10, which further comprises measuring aPTH peptide fragment level and/or total PTH level.
 31. The method ofclaim 30, wherein said sample is contacted with one or more isolatedantibodies, and wherein each of said one or more isolated antibodiesspecifically bind one or more PTH peptide fragments selected from thegroup consisting of: PTH₃₉₋₈₄, PTH₁₋₃₄, PTH₄₃₋₆₈, PTH₇₋₈₄, PTH₃₉₋₆₈,PTH₅₃₋₈₄, PTH₆₅₋₈₄, PTH₄₄₋₆₈, PTH₁₉₋₈₄, PTH₂₃₋₈₄, PTH₁₋₃₈, PTH₁₋₄₈,PTH₁₋₅₈, PTH₁₋₆₈, and PTH₁₋₇₈.
 32. The method of claim 30, which furthercomprises comparing at least two parameters selected from the groupconsisting of the whole PTH level, total PTH peptide fragment level,total PTH level, C-terminal PTH fragment (cPTH) level, N-terminal PTHfragment level, and mid-terminal PTH fragment (mPTH) level.
 33. Themethod of claim 32, wherein the results of said comparison are used todetermine whether the mammal suffers from a bone turnover relateddisorder.
 34. The method of claim 33, which is used in the diagnosis ormonitoring of treatment for adynamic bone disease.
 35. The method ofclaim 32, wherein the comparison is in the form of a ratio or proportionbetween the whole PTH level and the total PTH level.
 36. The method ofclaim 32, wherein the comparison is in the form of a ratio or proportionbetween the whole PTH level versus the combined total of the total PTHlevel minus the whole PTH level.
 37. The method of claim 10, which isused for differentiating between a person having substantially normalparathyroid function and having hyperparathyroidism.
 38. A kit formeasuring a physiological level of whole parathyroid hormone (PTH) in amammalian sample, which kit comprises, in a container, an isolatedantibody that specifically binds to an N-terminal sequence of whole(PTH) and is capable of detecting said whole PTH at a physiologicallevel in a mammalian sample, with a proviso that said isolated antibodyavoids binding to a non-(1-84) or non-(1-86) PTH fragment in saidsample, wherein said isolated antibody specifically binds to an epitopecomprised in PTH₁₋₅, PTH₁₋₆, PTH₁₋₇, PTH₁₋₈, PTH₁₋₉, PTH₁₋₁₀, PTH₁₋₁₁,PTH₁₋₁₂, PTH₁₋₁₃, PTH₁₋₁₄ or PTH₁₋₁₅.
 39. The method of claim 10,wherein the physiological level of whole PTH is from about 7 picogram/mlto about 39 picogram/ml.
 40. The method of claim 23, wherein the captureantibody is attached to the surface indirectly.
 41. The method of claim10, wherein the complex is assessed in a heterogeneous assay format. 42.The method of claim 10, wherein the isolated antibody avoids bindinggreater than about 90% of a non-whole PTH fragment in said sample. 43.The method of claim 10, wherein the isolated antibody specifically bindsto an epitope comprised in PTH₁₋₅ or PTH₁₋₆.
 44. The method of claim 22,wherein one of the first or the second antibody is labeled.
 45. A methodfor measuring a physiological level of whole parathyroid hormone (PTH)in a mammalian sample, which method comprises: a) obtaining a samplefrom a mammal to be tested; b) contacting said sample with an isolatedantibody that specifically binds to an N-terminal sequence of whole PTHand is capable of detecting said whole PTH at a physiological level insaid mammalian sample, with a proviso that said isolated antibody avoidsbinding to a non-whole PTH fragment; and c) assessing a complex formedbetween said whole PTH, if present in said sample, and said antibody, tomeasure physiological level of said whole PTH in said mammalian sample,wherein said antibody specifically binds to an epitope comprised inPTH₁₋₆, PTH₁₋₈, PTH₁₋₉, PTH₁₋₁₂, PTH₁₋₁₅, or PTH₃₋₁₂.
 46. The method ofclaim 45, wherein the non-whole PTH fragment is a peptide having anamino acid sequence of human PTH₇₋₈₄.
 47. A method for measuring aphysiological level of whole parathyroid hormone (PTH) in a mammaliansample, which method comprises: a) obtaining a sample from a mammal tobe tested; b) contacting said sample with an isolated antibody thatspecifically binds to an N-terminal sequence of whole PTH and is capableof detecting said whole PTH at a physiological level in said mammaliansample, with a proviso that said isolated antibody avoids binding to anon-whole PTH fragment; and c) assessing a complex formed between saidwhole PTH, if present in said sample, and said antibody, to measurephysiological level of said whole PTH in said mammalian sample, whereinsaid antibody specifically binds to the PTH peptide human PTH₁₋₈, ratPTH₁₋₈, mouse PTH₁₋₈, bovine PTH₁₋₈, canine PTH₁₋₈, porcine PTH₁₋₈,horse PTH₁₋₈, human PTH₁₋₁₅, rat PTH₁₋₁₅, mouse PTH₁₋₁₅, bovine PTH₁₋₁₅,canine PTH₁₋₁₅, porcine PTH₁₋₁₅, or horse PTH₁₋₁₅, wherein at least fouramino acids in said peptide sequence are part of a reactive portion withthe antibody.
 48. The method of claim 47, wherein the non-whole PTHfragment is a peptide having an amino acid sequence of human PTH₇₋₈₄.49. The method of claim 10, wherein the isolated antibody thatspecifically binds to an N-terminal sequence of whole PTH is produced byimmunizing a mammal with whole PTH, collecting the antibody from themammal and isolating the antibody by binding the antibody to an epitopecomprised in PTH₁₋₅, PTH₁₋₆, PTH₁₋₇, PTH₁₋₈, PTH₁₋₉, PTH₁₋₁₀, PTH₁₋₁₁,PTH₁₋₁₂, PTH₁₋₁₃, PTH₁₋₁₄ or PTH₁₋₁₅, and the isolated antibodyspecifically binds to whole PTH.
 50. The method of claim 49, wherein themammal is a goat.
 51. The method of claim 49, wherein the whole PTH ishuman whole PTH and the peptide is a human or rat PTH peptide.
 52. Themethod of claim 49, wherein the whole PTH is rat whole PTH and thepeptide is a human or rat PTH peptide.
 53. The method of claim 10,wherein the mammal is a rat.
 54. The method of claim 10, wherein theisolated antibody that specifically binds to an N-terminal sequence ofwhole PTH is produced by immunizing a mammal with whole PTH, collectingthe antibody from the mammal and isolating the antibody using a peptideselected from the group consisting of PTH₁₋₅, PTH₁₋₆, PTH₁₋₇, PTH₁₋₈,PTH₁₋₉, PTH₁₋₁₀, PTH₁₋₁₁, PTH₁₋₁₂, PTH₁₋₁₃, PTH₁₋₁₄ and PTH₁₋₁₅, and theisolated antibody specifically binds to whole PTH.
 55. The method ofclaim 32, wherein the results of said comparison are used to monitorbone disease or disorder related treatment.
 56. The isolated antibody orantibody fragment of claim 8, wherein the non-whole PTH fragment isselected from the group consisting of PTH₄₋₈₄, PTH₅₋₈₄, PTH₆₋₈₄,PTH₇₋₈₄, PTH₈₋₈₄, PTH₉₋₈₄, PTH₁₀₋₈₄, PTH₁₁₋₈₄, PTH₁₂₋₈₄, PTH₁₃₋₈₄,PTH₁₄₋₈₄, and PTH₁₅₋₈₄.
 57. The method of claim 19, wherein thenon-whole PTH fragment is selected from the group consisting of PTH₄₋₈₄,PTH₅₋₈₄, PTH₆₋₈₄, PTH₇₋₈₄, PTH₈₋₈₄, PTH₉₋₈₄, PTH₁₀₋₈₄, PTH₁₁₋₈₄,PTH₁₂₋₈₄, PTH₁₃₋₈₄, PTH₁₄₋₈₄ and PTH₁₅₋₈₄.
 58. The isolated antibody ofclaim 1, which is a polyclonal antibody that specifically binds to wholePTH.
 59. The isolated antibody of claim 1, which is an antibody fragmentthat specifically binds to whole PTH.
 60. The isolated antibody of claim1, which specifically binds to an epitope comprised in PTH₁₋₆.
 61. Theisolated antibody of claim 1, which specifically binds to an epitopecomprised in PTH₁₋₇.
 62. The isolated antibody of claim 1, whichspecifically binds to an epitope comprised in PTH₁₋₈.
 63. The isolatedantibody of claim 1, which specifically binds to an epitope comprised inPTH₁₋₉.
 64. The isolated antibody of claim 1, which specifically bindsto an epitope comprised in PTH₁₋₁₀.
 65. The isolated antibody of claim1, which specifically binds to an epitope comprised in PTH₁₋₁₁.
 66. Theisolated antibody of claim 1, which specifically binds to an epitopecomprised in PTH₁₋₁₂.
 67. The isolated antibody of claim 1, whichspecifically binds to an epitope comprised in PTH₁₋₁₃.
 68. The isolatedantibody of claim 1, which specifically binds to an epitope comprised inPTH₁₋₁₄.
 69. The isolated antibody of claim 1, which specifically bindsto an epitope comprised in PTH₁₋₁₅.
 70. The isolated antibody of claim1, which specifically binds to an epitope comprised in PTH₃₋₁₂.
 71. Theisolated antibody of claim 1, which specifically binds to theparathyroid hormone peptide human PTH₁₋₈, wherein at least four aminoacids in said peptide sequence are part of a reactive portion with theantibody.
 72. The isolated antibody of claim 1, which specifically bindsto the parathyroid hormone peptide rat PTH₁₋₈, wherein at least fouramino acids in said peptide sequence are part of a reactive portion withthe antibody.
 73. The isolated antibody of claim 1, which specificallybinds to the parathyroid hormone peptide human PTH₁₋₁₂, wherein at leastfour amino acids in said peptide sequence are part of a reactive portionwith the antibody.
 74. The isolated antibody of claim 1, whichspecifically binds to the parathyroid hormone peptide rat PTH₁₋₁₂,wherein at least four amino acids in said peptide sequence are part of areactive portion with the antibody.
 75. The isolated antibody of claim1, wherein the isolated antibody avoids binding greater than about 90%of a non-whole PTH fragment in the mammalian sample.
 76. The isolatedantibody of claim 1, wherein the isolated antibody avoids bindinggreater than about 95% of a non-whole PTH fragment in the mammaliansample.
 77. The isolated antibody of claim 1, wherein the isolatedantibody avoids binding greater than about 99% or more of a non-wholePTH fragment in the mammalian sample.
 78. The method of claim 10,wherein the antibody is a polyclonal antibody that specifically binds towhole PTH.
 79. The method of claim 10, wherein the antibody is anantibody fragment that specifically binds to whole PTH.
 80. The methodof claim 10, wherein the complex is assessed by a competitive assayformat.
 81. The method of claim 33, which is used in the diagnosis ormonitoring of treatment for severe hyperparathyroidism.
 82. The methodof claim 10, which is used for monitoring parathyroid related bonedisease and treatment.
 83. The method of claim 10, which is used formonitoring effects of therapeutic treatment for hyperparathyroidism. 84.The method of claim 10, which is used for diagnosing parathyroid relatedbone disease.
 85. The method of claim 10, wherein the isolated antibodyspecifically binds to an epitope comprised in PTH₁₋₆.
 86. The method ofclaim 10, wherein the isolated antibody specifically binds to an epitopecomprised in PTH₁₋₇.
 87. The method of claim 10, wherein the isolatedantibody specifically binds to an epitope comprised in PTH₁₋₈.
 88. Themethod of claim 10, wherein the isolated antibody specifically binds toan epitope comprised in PTH₁₋₉.
 89. The method of claim 10, wherein theisolated antibody specifically binds to an epitope comprised in PTH₁₋₁₀.90. The method of claim 10, wherein the isolated antibody specificallybinds to an epitope comprised in PTH₁₋₁₁.
 91. The method of claim 10,wherein the isolated antibody specifically binds to an epitope comprisedin PTH₁₋₁₂.
 92. The method of claim 10, wherein the isolated antibodyspecifically binds to an epitope comprised in PTH₁₋₁₃.
 93. The method ofclaim 10, wherein the isolated antibody specifically binds to an epitopecomprised in PTH₁₋₁₄.
 94. The method of claim 10, wherein the isolatedantibody specifically binds to an epitope comprised in PTH₁₋₁₅.
 95. Themethod of claim 10, wherein the isolated antibody avoids binding greaterthan about 95% of a non-whole PTH fragment in the mammalian sample. 96.The method of claim 10, wherein the isolated antibody avoids bindinggreater than about 99% or more of a non-whole PTH fragment in themammalian sample.
 97. The isolated antibody of claim 1, whichspecifically binds to at least four amino acids in PTH₁₋₈ sequence. 98.The isolated antibody of claim 97, which avoids binding to PTH₇₋₈₄fragment.
 99. The isolated antibody of claim 1, which specifically bindsto PTH₁₋₆ sequence as part of whole PTH.
 100. The isolated antibody ofclaim 1, which specifically binds to PTH₁₋₇ sequence as part of wholePTH.
 101. The isolated antibody of claim 1, which specifically binds toPTH₁₋₈ sequence as part of whole PTH.
 102. The isolated antibody ofclaim 1, which specifically binds to PTH₁₋₉ sequence as part of wholePTH.
 103. The isolated antibody of claim 1, which specifically binds toPTH₁₋₁₀ sequence as part of whole PTH.
 104. The isolated antibody ofclaim 1, which specifically binds to PTH₁₋₁₁ sequence as part of wholePTH.
 105. The isolated antibody of claim 1, which specifically binds toPTH₁₋₁₂ sequence as part of whole PTH.
 106. The isolated antibody ofclaim 1, which specifically binds to PTH₁₋₁₃ sequence as part of wholePTH.
 107. The isolated antibody of claim 1, which specifically binds toPTH₁₋₁₄ sequence as part of whole PTH.
 108. The isolated antibody ofclaim 1, which specifically binds to PTH₁₋₁₅ sequence as part of wholePTH.
 109. A method for measuring a physiological level of wholeparathyroid hormone (PTH) in a mammalian sample, which method comprises:a) obtaining a sample from a mammal to be tested; b) contacting saidsample with an isolated antibody that specifically binds to anN-terminal sequence of whole PTH and is capable of detecting said wholePTH at a physiological level in said mammalian sample, with a provisothat said isolated antibody avoids binding to a non-whole PTH fragmentin said sample; and c) assessing a complex formed between said wholePTH, if present in said sample, and said antibody, to measurephysiological level of said whole PTH in said mammalian sample, whereinsaid isolated antibody specifically binds to PTH₁₋₆ sequence as part ofwhole PTH, PTH₁₋₇ sequence as part of whole PTH, PTH₁₋₈ sequence as partof whole PTH, PTH₁₋₉ sequence as part of whole PTH, PTH₁₋₁₀ sequence aspart of whole PTH, PTH₁₋₁₁ sequence as part of whole PTH, PTH₁₋₁₂sequence as part of whole PTH, PTH₁₋₁₃ sequence as part of whole PTH,PTH₁₋₁₄ sequence as part of whole PTH, or PTH₁₋₁₅ sequence as part ofwhole PTH.
 110. The method of claim 109, wherein the isolated antibodyavoids binding to PTH₇₋₈₄ fragment.
 111. The method of claim 109,wherein the isolated antibody specifically binds to PTH₁₋₆ sequence aspart of whole PTH.
 112. The method of claim 109, wherein the isolatedantibody specifically binds to PTH₁₋₇ sequence as part of whole PTH.113. The method of claim 109, wherein the isolated antibody specificallybinds to PTH₁₋₈ sequence as part of whole PTH.
 114. The method of claim109, wherein the isolated antibody specifically binds to PTH₁₋₉ sequenceas part of whole PTH.
 115. The method of claim 109, wherein the isolatedantibody specifically binds to PTH₁₋₁₀ sequence as part of whole PTH.116. The method of claim 109, wherein the isolated antibody specificallybinds to PTH₁₋₁₁ sequence as part of whole PTH.
 117. The method of claim109, wherein the isolated antibody specifically binds to PTH₁₋₁₂sequence as part of whole PTH.
 118. The method of claim 109, wherein theisolated antibody specifically binds to PTH₁₋₁₃ sequence as part ofwhole PTH.
 119. The method of claim 109, wherein the isolated antibodyspecifically binds to PTH₁₋₁₄ sequence as part of whole PTH.
 120. Themethod of claim 109, wherein the isolated antibody specifically binds toPTH₁₋₁₅ sequence as part of whole PTH.
 121. A kit for measuring aphysiological level of whole parathyroid hormone (PTH) in a mammaliansample, which kit comprises, in a container, an isolated antibody thatspecifically binds to an N-terminal sequence of whole PTH and is capableof detecting said whole PTH at a physiological level in a mammaliansample, with a proviso that said isolated antibody avoids binding to anon-whole PTH fragment in said sample, wherein said isolated antibodyspecifically binds to PTH₁₋₆ sequence as part of whole PTH, PTH₁₋₇sequence as part of whole PTH, PTH₁₋₈ sequence as part of whole PTH,PTH₁₋₉ sequence as part of whole PTH, PTH₁₋₁₀ sequence as part of wholePTH, PTH₁₋₁₁ sequence as part of whole PTH, PTH₁₋₁₂ sequence as part ofwhole PTH, PTH₁₋₁₃ sequence as part of whole PTH, PTH₁₋₁₄ sequence aspart of whole PTH, or PTH₁₋₁₅ sequence as part of whole PTH.
 122. Thekit of claim 121, wherein the isolated antibody avoids binding toPTH₇₋₈₄ fragment.